Recombinant infectious laryngotracheitis virus and uses thereof

ABSTRACT

Recombinant, attenuated infectious laryngotracheitis virus containing a deletion or deletion in a glycoprotein gene are provided. Also provided are glycoprotein genes, gD, gI, gG, and gE as well as other viral genes.

This application is a division of application U.S. Ser. No. 08/468,190filed Jun. 6, 1995 now abandoned which is a continuation of U.S. Ser.No. 08/410,121 filed Mar. 23, 1995 now abandoned which is acontinuation-in-part of U.S. Ser. No. 08/126,597 filed Sep. 24, 1993 nowabandoned.

Within this application several publications are referenced by arabicnumerals within parentheses. Full citations for these publications maybe found at the end of the specification preceding the claims. Thedisclosures of these publications are hereby incorporated by referenceinto this application in order to more fully describe the state of theart to which this invention pertains.

BACKGROUND OF THE INVENTION

Infectious laryngotracheitis virus is a herpesvirus that causes arespiratory illness of varying virulence in chickens. Live attenuatedILTV vaccines are available to protect against the disease, but severalreports have implicated vaccine viruses in the possible recurrence andspread of the disease (65 and 72), limiting vaccination to use inuninfected birds early in an outbreak. In order to design a moreefficacious, attenuated vaccine, the genomic organization of the ILTVvirus has been studied.

The ability to isolate viral DNA and clone this isolated DNA intobacterial plasmids has greatly expanded the approaches available to makeviral vaccines. The methods used to make the present invention involvemodifying cloned viral DNA sequences by insertions, deletions and singleor multiple base changes. The modified DNA is then reinserted into theviral genome to render the virus non-pathogenic. The resulting livevirus may then be used in a vaccine to elicit an immune response in ahost animal and to protect the animal against a disease.

One group of animal viruses, the herpesviruses or Herpetoviridae, is anexample of a class of viruses amenable to this approach. These virusescontain 100,000 to 200,000 base pairs of DNA as their genetic material.Importantly several regions of the genome have been identified that arenonessential for the replication of virus in vitro in cell culture.Modifications in these regions of the DNA may lower the pathogenicity ofthe virus, i.e., attenuate the virus. For example, inactivation of thethymidine kinase gene renders human herpes simplex virus non-pathogenic(1), and pseudorabies virus of swine non-pathogenic (2).

Removal of part of the repeat region renders human herpes simplex virusnon-pathogenic (3, 4). A repeat region has been identified in Marek'sdisease virus that is associated with viral oncogenicity (5). A regionin herpesvirus saimiri has similarly been correlated with oncogenicity(6). Removal of part of the repeat region renders pseudorabies virusnon-pathogenic (U.S. Pat. No. 4,877,737, issued Oct. 31, 1989). A regionin pseudorabies virus has been shown to be deleted innaturally-occurring vaccine strains (7, 8) and it has been shown thatthese deletions are at least partly responsible for the lack ofpathogenicity of these strains.

It is generally agreed that herpesviruses contain non-essential regionsof DNA in various parts of the genome. Some of these regions areassociated with virulence of the virus, and modification of them leadsto a less-pathogenic virus, from which a vaccine may be derived.

Infectious laryngotracheitis virus (ILTV), an alpha herpesvirus (9), isan important pathogen of poultry in the USA, Europe, and Australia,responsible for egg production losses and death (10). It causes an acutedisease of chickens which is characterized by respiratory depression,gasping and expectoration of bloody exudate. Viral replication islimited to cells of the respiratory tract wherein infection of thetrachea gives rise to tissue erosion and hemorrhage.

In chickens, no drug has been effective in reducing the degree of lesionformation or in decreasing clinical signs. Vaccination of birds withvarious modified forms of the ILT virus derived by cell passage and/ortedious regimes of administration have been used to confer acceptableprotection in susceptible chickens. Due to the limited degree ofattenuation of current ILTV vaccines care must be taken to assure thatthe correct level of virus is maintained, enough to provide protection,but not enough to cause disease in the flock (11-21). Furthermore, theseviruses may revert back to virulence. causing disease rather thanproviding protection against it.

ILTV has been analyzed at the molecular level. Restriction maps of theILTV genome have been reported (22-26). The DNA sequence of severalgenes have been identified, i.e., thymidine kinase (27, 28),glycoprotein gB (27, 29, 30), ribonucleotide reductase (27, 31), capsidp40 (31, 32).

Furthermore, Shepard, et al. (53) disclosed that several genes locatedin the unique long region of the infectious laryngotracheitis virusgenomic DNA are non-essential for viral replication.

Applicants have unexpectedly found that the unique short region of theILT virus genomic DNA contains genes that are associated with ILTVvirulence and that a deletion in those genes leads to an attenuatedILTV. Particularly, it was found that a deletion in the glycoprotein G(gG) gene of the ILT virus results in an attenuated virus, which isuseful as a vaccine against subsequent attack by a virulent ILTVstrains.

Applicants also found that a deletion in the glycoprotein I (gI) gene ofthe unique short region also attenuates the ILTV. Furthermore, it iscontemplated that a deletion in the US2 gene, the UL-47 like gene, andthe glycoprotein g60 gene of the unique short region will also attenuatethe ILTV.

ILTV can become latent in healthy animals which makes them potentialcarriers of the virus. For this reason, it is clearly advantageous to beable to distinguish animals vaccinated with non-virulent virus fromanimals infected with disease-causing wild-type or naturally-occurringvirus. The development of differential vaccines and companion diagnostictests has proven valuable in the management of pseudorabies disease(55). A similar differential marker vaccine would be of great value inthe management of ILTV caused disease. The construction of differentialdiagnostics has focused on the deletion of glycoprotein. Theoretically,the glycoprotein chosen to be the diagnostic marker should have thefollowing characteristics: (1) the glycoprotein and its gene should benon-essential for the production of infectious virus in tissue culture:(2) the glycoprotein should elicit a major serological response in theanimal; and (3) the glycoprotein should not be one that makes asignificant contribution to the protective immunity.

The ILT virus has been shown to specify at least four majorglycoproteins as identified by monoclonal antibodies (M_(r)=205K, 115K,90K and 60K). Three glycoproteins seem to be antigenically related(M_(r)=205K, 115K, and 90K) (34-36).

Three major ILT virus glycoproteins, gB (29, 30), gC (27, 51), and g60(34, 53) have been described in the literature. These three genes havebeen sequenced and two of the ILTV genes have been shown to behomologous to the HSV glycoproteins gB, and gC.

Of these, it is known that the ILTV gB gene is an essential gene andwould not be appropriate as deletion marker genes. Furthermore, the gCgene of herpesviruses has been shown to make a significant contributionto protective immunity as a target of neutralizing antibody (56) and asa target of cell-mediated immunity (57). Therefore, the gC gene is notdesirable as a deletion marker gene.

As to other glycoprotein encoding genes cited above, it is not knownwhether or not they would be suitable candidates for deletion in orderto construct a recombinant ILT virus which can be used as a diagnosticvaccine.

Applicants have unexpectedly found that there are two glycoproteinencoding genes located within the unique short region of the ILT viralgenome which could be safely deleted in order to construct a recombinantILT virus that can be used as a diagnostic vaccine. These are theglycoprotein gG gene and the glycoprotein gI gene. By geneticallyengineering an ILT virus with a deletion in the glycoprotein G gene orthe glycoprotein I gene, a ILT virus is produced which does not expressany glycoprotein G or glycoprotein I. None of the prior arts teach orsuggest that these two genes in the unique short region of the virus areappropriate candidates for deletion in order to create a diagnostic ILTvirus vaccine. Although several of the herpesviruses have beengenetically engineered, no examples of recombinant ILTV have beenreported.

The ability to engineer DNA viruses with large genomes, such as vacciniavirus and the herpesviruses, has led to the finding that theserecombinant viruses can be used as vectors to deliver vaccine antigensand therapeutic agents for animals. The herpesviruses are attractivecandidates for development as vectors because their host range isprimarily limited to a single target species (37) and they have thecapacity for establishing latent infection (38) that could provide forstable in vivo expression of a foreign gene. Although severalherpesvirus species have been engineered to express foreign geneproducts, recombinant infectious laryngotracheitis viruses expressingforeign gene products have not been constructed. The infectiouslaryngotracheitis viruses described above may be used as vectors for thedelivery of vaccine antigens from microorganisms causing importantpoultry diseases. Other viral antigens which may be included in amultivalent vaccine with an ILTV vector include infectious bronchitisvirus (IBV), Newcastle disease virus (NDV), infectious bursal diseasevirus (IBDV), and Marek's disease virus (MDV). Such multivalentrecombinant viruses would protect against ILT disease as well as otherdiseases. Similarly the infectious laryngotracheitis viruses may be usedas vectors for the delivery of therapeutic agents. The therapeutic agentthat is delivered by a viral vector of the present invention must be abiological molecule that is a by-product of ILTV replication. Thislimits the therapeutic agent in the first analysis to either DNA. RNA orprotein. There are examples of therapeutic agents from each of theseclasses of compounds in the form of anti-sense DNA. anti-sense RNA (39).ribozymes (40), suppressor tRNAs (41), interferon-inducing doublestranded RNA and numerous examples of protein therapeutics, fromhormones, e.g. insulin, to lymphokines, e.g., interferons andinterleukins, to natural opiates. The discovery of these therapeuticagents and the elucidation of their structure and function does notnecessarily allow one to use them in a viral vector delivery system,however, because of the experimentation necessary to determine whetheran appropriate insertion site exists.

ILTV is classified as an alpha herpesvirus with a type D genome (78)composed of a unique long region and a unique short region flanked byinverted repeats. A genomic restriction map of an Australian ILTVisolate (SA-2) was described by Johnson et al. (66). Using this map, Guoet al. (62) isolated and sequenced a DNA fragment from the USDAchallenge strain which appeared to be derived from the unique shortregion. Applicants map the USDA challenge strain of ILTV, and reportscharacteristics of the putative genes present in the unique shortregion. The map disclosed herewith indicates that the sequenceidentified by Guo et al. (62) is part of the short repeat sequence, andis not from the unique short. Other reports (69 and 70) describe thesequences of two genes, one homologous to PRV gG and the other unlikeother reported herpesvirus genes. These two genes were mapped to theunique long region of SA-2. However, these sequences are identical tosequences identified in this application as being from the unique shortregion. The data in this application indicate that the overallorganization of the short region of ILTV is similar to otherherpesviruses.

SUMMARY OF THE INVENTION

The present invention provides a recombinant, attenuatedinfectiouslaryngotracheitis virus comprising the infectiouslaryngotracheitis viral genome which contains a deletion in theglycoprotein gG gene. This attenuated virus is useful as a vaccineagainst infectious laryngotracheitis virus.

The present invention also provides a recombinant, attenuated infectiouslaryngotracheitis virus comprising the infectious laryngotracheitisviral genome which contains a deletion in the US2 gene, UL47-like gene,virus4 gene or glycoprotein g60 gene.

The present invention also provides a method for distinguishing chickensor other poultry vaccinated with a recombinant infectiouslaryngotracheitis virus which produces no glycoprotein gG from thoseinfected with a naturally-occuring infectious laryngotracheitis virus.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1H:

The nucleotide sequence of 13,473 base pairs of contiguous DNA from theunique short region of the ILT virus. This sequence contains the entire13,098 base pair unique short region as well as 273 base pairs of repeatregion at one end and 102 base pairs of repeat region at the other end.The nucleotide sequences of FIGS. 1A-1H begin with the internal repeatsequence and end within the terminal repeat sequence. The unique shortregion begins at base pair 274 of this Figure. Sequence ID NO:59contains the nucleotide sequence of 18,912 base pares of contiguous DNAfrom the unique short and repeat regions of the ILT virus. This sequencecontains the entire 13,094 base pair unique short region as well as 2909base paris of internal repeat region and 2909 base paris of shortterminal repeat region. The nucleotide sequences begin with the internalrepeat sequence and end within the terminal repeat sequence. The uniqueshort region begins at base pair 2910.

FIG. 2:

Asp718 I restriction enzyme map of the infectious laryngotracheitisvirus (ILTV) USDA 83-2 genome. The upper diagram identifies the uniquelong (U_(L)), internal repeat (IR), unique short (U_(S)), and terminalrepeat (TR) sections found in the ILTV genome. A map of the Asp718 Irestriction endonuclease sites in the ILTV genome is shown below.Letters A through O identify Asp718 I restriction endonuclease fragmentswith “A” representing the largest fragment. Fragment “L” is the 2.5 kbAsp718 I fragment, fragment “H” is the 5164 bp Asp718 I fragment, andfragment “G” is the 8.0 kb Asp718 I fragment. The fragments marked withasterisks contain a hypervariable region of approximately 900 bp that isrepeated from one to 12 times. Since no one size predominates, thesefragments appear in submolar amounts that are not well resolved on anethidium bromide stained gel. The position of these repeats is indicatedin the Figures by the crooked dashed lines.

FIG. 3:

Open reading frames within the unique short region of infectiouslaryngotracheitis virus (ILTV) USDA 83-2. The 13,473 base pairs of theshort region of ILTV contains the entire 13,098 base pair unique shortregion as well as 273 base pairs of repeat region at one end and 102base pairs of repeat region at the other end. The unique short regioncontains 13 methionine initiated open reading frames (ORF) of greaterthan or equal to 110 amino acids (excluding smaller nested ORFs). All 13ORFs were aligned to the Entrez release 6.0 virus division of theGenbank DNA database utilizing the IBI MacVector Protein to DNAalignment option (default settings). Eight of the ORFs exhibitedsignificant homology to one or more other virus genes: unique short(US2), protein kinase (PK), unique long 47-like (UL47-like), andglycoproteins gG, g60, gD, gI, and gE.

FIGS. 4A-4B:

Detailed description of the DNA insertion in Homology Vector 472-73.27.Diagram showing the orientation of DNA fragments assembled in plasmid472-73.27. The origin of each fragment is indicated in the table. Thesequences located at each of the junctions between fragments are alsoshown (SEQ ID NO's: 20, 21, 22 and 23). The restriction sites used togenerate each fragment as well as the synthetic linker sequences whichwere used to join the fragments are described for each junction. Thelocation of several gene coding regions and regulatory elements is alsogiven. Restriction sites in brackets [ ] indicate the remnants of siteswhich were destroyed during construction. The following abbreviationsare used, infectious laryngotracheitis virus (ILTV), humancytomegalovirus immediate early (HCMV IE), pseudorabies virus (PRV),lactose operon Z gene (lacZ), Escherichia coli (E. coli),polyadenylation signal (poly A), and base pairs (BP).

FIGS. 5A-5B:

Detailed description of the DNA insertion in Homology Vector 501-94.Diagram showing the orientation of DNA fragments assembled in plasmid501-94. The origin of each fragment is indicated in the table. Thesequences located at each of the junctions between fragments are alsoshown (SEQ ID NO's: 24, 25, 26, and 27). The restriction sites used togenerate each fragment as well as the synthetic linker sequences whichwere used to join the fragments are described for each junction. Thelocation of several gene coding regions and regulatory elements is alsogiven. Restriction sites in brackets [ ] indicate the remnants of siteswhich were destroyed during construction.

The following abbreviations are used, infectious laryngotracheitis virus(ILTV), human cytomegalovirus immediate early (HCMV IE), pseudorabiesvirus (PRV), lactose operon Z gene (lacZ), Escherichia coli (E. coli),polyadenylation signal (poly A), thymidine kinase (TK), and base pairs(BP).

FIGS. 6A-6B:

Detailed description of the DNA insertion in Homology Vector 544-55.12.Diagram showing the orientation of DNA fragments assembled in plasmid544-55.12. The origin of each fragment is indicated in the table. Thesequences located at each of the junctions between fragments are alsoshown (SEQ ID NO's: 28, 29, 30, and 31). The restriction sites used togenerate each fragment as well as the synthetic linker sequences whichwere used to join the fragments are described for each junction. Thelocation of several gene coding regions and regulatory elements is alsogiven. Restriction sites in brackets [ ] indicate the remnants of siteswhich were destroyed during construction. The following abbreviationsare used, infectious laryngotracheitis virus (ILTV), herpes simplexvirus type 1 (HSV-1), pseudorabies virus (PRV), β-glucuronidase gene(uidA), Escherichia coli (E. coli), polyadenylation signal (poly A), andbase pairs (BP).

FIGS. 7A-7C:

Detailed description of the DNA insertion in Homology Vector 562-61.1F.Diagram showing the orientation of DNA fragments assembled in plasmid562-61.1F. The origin of each fragment is indicated in the table. Thesequences located at each of the junctions between fragments are alsoshown (SEQ ID NO's: 32, 33, 34 35, 36 and 37). The restriction sitesused to generate each fragment as well as the synthetic linker sequenceswhich were used to join the fragments are described for each junction.The location of several gene coding regions and regulatory elements isalso given. Restriction sites in brackets [ ] indicate the remnants ofsites which were destroyed during construction. The followingabbreviations are used, infectious laryngotracheitis virus (ILTV),herpes simplex virus type 1 (HSV-1), pseudorabies virus (PRV),β-glucuronidase gene (uidA), Escherichia coli (E. coli), polyadenylationsignal (poly A), and base pairs (BP).

FIGS. 8A-8C:

Detailed description of the DNA insertion in Homology Vector 560-52.F1.Diagram showing the orientation of DNA fragments assembled in plasmid560-52.F1. The origin of each fragment is indicated in the table. Thesequences located at each of the junctions between fragments are alsoshown (SEQ ID NO's: 38, 39, 40, 41, and 42). The restriction sites usedto generate each fragment as well as the synthetic linker sequenceswhich were used to join the fragments are described for each junction.The location of several gene coding regions and regulatory elements isalso given. Restriction sites in brackets [ ] indicate the remnants ofsites which were destroyed during construction. The followingabbreviations are used, infectious laryngotracheitis virus (ILTV),herpes simplex virus type 1 (HSV-1), pseudorabies virus (PRV),β-glucuronidase gene (uidA), Escherichia coli (E. coli), polyadenylationsignal (poly A), unique long 47 (UL47-like), open reading frame 4(ORF4), glycoprotein G (gG), and base pairs (BP).

FIGS. 9A-9B:

Detailed description of the DNA insertion in Homology Vector 579-14.G2.Diagram showing the orientation of DNA fragments assembled in plasmid579-14.G2. The origin of each fragment is indicated in the table. Thesequences located at each of the junctions between fragments are alsoshown (SEQ ID NO's: 43, 44, 45, and 46). The restriction sites used togenerate each fragment as well as the synthetic linker sequences whichwere used to join the fragments are described for each junction. Thelocation of several gene coding regions and regulatory elements is alsogiven. Restriction sites in brackets [ ] indicate the remnants of siteswhich were destroyed during construction. The following abbreviationsare used, infectious laryngotracheitis virus (ILTV), herpes simplexvirus type 1 (HSV-1), pseudorabies virus (PRV), β-glucuronidase gene(uidA), Escherichia coli (E. coli), polyadenylation signal (poly A), andbase pairs (BP).

FIGS. 10A-10B:

Detailed description of the DNA insertion in Plasmid Vector 544-39.13.Diagram showing the orientation of DNA fragments assembled in plasmid544-39.13. The origin of each fragment is indicated in the table. Thesequences located at each of the junctions between fragments are alsoshown (SEQ ID NO's: 47, 48, 49, and 50). The restriction sites used togenerate each fragment as well as the synthetic linker sequences whichwere used to join the fragments are described for each junction. Thesynthetic linker sequences are underlined by a heavy bar. The locationof several gene coding regions and regulatory elements is also given.Restriction sites in brackets [ ] indicate the remnants of sites whichwere destroyed during construction. The following abbreviations areused, pseudorabies virus (PRV), β-glucuronidase gene (uidA), Escherichiacoli (E. coli), herpes simplex virus type 1 (HSV-1), polyadenylationsignal (poly A), and base pairs (BP).

FIGS. 11A-11C:

Detailed description of the DNA insertion in Plasmid Vector 388-65.2.Diagram showing the orientation of DNA fragments assembled in plasmid388-65.2. The origin of each fragment is indicated in the table. Thesequences located at each of the junctions between fragments are alsoshown (SEQ ID NO's: 51, 52, 53, and 54). The restriction sites used togenerate each fragment as well as the synthetic linker sequences whichwere used to join the fragments are described for each junction. Thesynthetic linker sequences are underlined by a heavy bar. The locationof several gene coding regions and regulatory elements is also given.Restriction sites in brackets [ ] indicate the remnants of sites whichwere destroyed during construction. The following abbreviations are usedhuman cytomegalovirus immediate early (HCMV IE), lactose operon Z gene(lacZ). Escherichia coli (E. coli), pseudorabies virus (PRV),polyadenylation signal (poly A), and base pairs (BP).

FIG. 12:

The genome of the ILTV virus, identifying the unique long (U_(L)),unique short (US), internal repeat (IR), and terminal repeat (TR) isshown. The BamHI, Asp718I, NotI, and SfiI restriction maps of the virusare drawn underneath, with the highly repetitive region of the shortrepeats indicated by a set of wavy lines. The position of the cosmidsused to determine the map of ILTV are drawn beneath the restriction map.Note that cosmid 2F12 contains two non-contiguous sections. Three probesused to characterize the ILTV genome are indicated as P1, P2, and P3. P1is a 0.9 kb NotI fragment found at the terminus of the unique longregion, P2 is the 856 bp HindIII fragment found in multiple copieswithin the short repeat, and P3 is a 6.6 kb NotI fragment used toidentify the fragments at the end of the terminal repeat.

FIG. 13:

The region sequenced, and the positions of the Asp718I, BamHI, NotI, andSfiI sites are shown. The and extent and orientation of the open readingframes found in the ILTV unique short and the flanking short repeatregions are indicated.

FIG. 14:

Southern blot showing the repetition of an 856 bp element within theshort repeat. Genomic ILTV DNA digested with SfiI (a), HindIII (b), NotI(c), Asp718I (d), or BamHI (e) was probed with an 856 bp HindIIIfragment from the short repeat. Positions of molecular weight markersare indicated.

FIG. 15:

Depiction of the position of the 856 bp repeat region in the USDAstrain, compared to the same region from the SA-2 strain as described byJohnson et al. Three repeats are arbitrarily shown in the USDA strain,the region is not repeated in SA2. B=BarnHI, H=HindIII, R=856 bp repeat.

FIG. 16:

Southern blot identifying fragments from the internal and terminalrepeat that hybridized to a 6.6 kb NotI fragment containing the junctionof the unique long and the internal repeat. Genomic ILTV DNA digestedwith NotI (a), Asp718I (b), and BamHI (c) was probed with the 6.6 kbNotI fragment. Positions of molecular weight markers are indicated.

FIG. 17:

The relationship of herpesvirus UL47 proteins to each other and to theILTV UL47 homolog in a conserved region. Amino acids shared between ILTVUL47 and the other UL47 proteins are in boldface type. Pairwisecomparisons have been made between the sequences as shown. A verticalbar indicates an identical amino acid, two dots indicate a positiveprobable acceptable mutation rate and one dot indicates a neutralprobable acceptable mutation rate (60).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a recombinant infectiouslaryngotracheitis virus comprising the infectious laryngotracheitisviral genome which contains a deletion in the unique short region of theviral genome, wherein the deletion is in the glycoprotein gG gene. Saiddeletion attenuates the virus, rendering it suitable for use as avaccine against infectious laryngotracheitis virus. A preferredembodiment of this invention is a recombinant infectiouslaryngotracheitis designated S-ILT-014 (ATCC Accession No. 2427). TheS-ILT-014 virus has been deposited pursuant to the Budapest Treaty onthe International Deposit of Microorganisms for the Purposes of PatentProcedure with the Patent Culture Depository of the American TypeCulture Collection. 12301 Parklawn Drive, Rockville, Md. 20852 U.S.A. onSep. 22, 1993 under ATCC Accession No. 2427). Another preferredembodiment of this invention is a recombinant infectiouslaryngotracheitis virus designated S-ILT-002.

For purposes of this invention, “a recombinant infectiouslaryngotracheitis virus” is a live infectious laryngotracheitis viruswhich has been generated by the recombinant methods well known to thoseof skill in the art, e.g., the methods set forth in DNA TRANSFECTION FORGENERATING RECOMBINANT ILT VIRUS in Materials and Methods, and the virushas not had genetic material essential for the replication of theinfectious laryngotracheitis virus deleted.

The present invention further provides a recombinant infectiouslaryngotracheitis virus comprising the infectious laryngotracheitisviral genome which contains a deletion in the glycoprotein gG gene and adeletion in the US2 gene. One preferred embodiment of this invention isa recombinant infectious laryngotracheitis virus designated S-ILT-009.

The present invention further provides a recombinant laryngotracheitisvirus comprising the infectious laryngotracheitis viral genome whichcontains a deletion in the glycoprotein gG gene and a deletion in theORF4 gene.

The present invention further provides a recombinant infectiouslaryngotracheitis virus which comprises the infectious laryngotracheitisviral genome which contains a deletion in the glycoprotein gG gene and adeletion in the UL47-like gene.

The present invention further provides a recombinant infectiouslaryngotracheitis virus which comprises the infectious laryngotracheitisviral genome which contains a deletion in the glycoprotein gG gene, adeletion in the ORF4 gene, and a deletion in the UL47-like gene. Apreferred embodiment of this invention is a recombinant infectiouslaryngotracheitis virus designated S-ILT-015.

The present invention further provides a recombinant infectiouslaryngotracheitis virus which comprises the infectious laryngotracheitisviral genome which contains a deletion in the glycoprotein gG gene and adeletion in the glycoprotein g60 gene. A preferred embodiment of thisinvention is a recombinant infectious laryngotracheitis virus designatedS-ILT-017.

The present invention further provides a recombinant infectiouslaryngotracheitis virus comprising the infectious laryngotracheitisviral genome which contains a deletion in the glycoprotein gG gene and adeletion in the glycoprotein gI gene.

The present invention further provides a recombinant infectiouslaryngotracheitis virus which comprises the infectious laryngotracheitisviral genome containing a deletion in the glycoprotein gG gene and adeletion in the thymidine kinase (TK) gene.

The present invention further provides a recombinant infectiouslaryngotracheitis virus comprising the infectious laryngotracheitisvirus genome which contains a deletion in the unique short region of theviral genome, wherein the deletion in the glycoprotein gG gene, andwhich also contains an insertion of a foreign gene. The foreign gene isinserted into a non-essential site of the infectious laryngotracheitisviral genome in such a way that it is capable of being expressed in arecombinant infectious laryngotracheitis infected host cell.

For purposes of this invention, “a non-essential site” of the infectiouslaryngotracheitis viral genome is a region of the viral genome which isnot necessary for viral infection and replication.

The following non-essential sites of the infectious laryngotracheitisviral genome are preferred sites for inserting a foreign gene into thevirus: the thymidine kinase (TK) gene, the US2 gene, the UL47-like gene,the ORF4 gene, the glycoprotein gG gene, the glycoprotein g60 gene, andthe glycoprotein gI gene.

The foreign gene, which is inserted into a non-essential site in theinfectious laryngotracheitis viral genome, may encode a screenablemarker, such as E. coli B-galactosidase or E. coli B-glucuronidase.

The foreign gene which is inserted into a non-essential site in theinfectious laryngotracheitis viral genome, may encode an antigenicpolypeptide which, when introduced into the host cell, inducesproduction of protective antibodies against an avian disease causingagent from which the antigen is derived or derivable. Antigenicpolypeptide which includes, but is not limited to: marek's disease virus(MDV) gA, marek's disease virus gB, marek's disease virus gD, Newcastledisease virus (NDV) HN, Newcastle disease virus F, infectiouslaryngotracheitis virus (ILT) gB, infectious laryngotracheitis virus gI,infectious laryngotracheitis virus gD, infectious bursal disease virus(IBDV) VP2, infectious bursal disease virus VP3, infectious bursaldisease virus VP4, infectious bursal disease virus polyprotein,infectious bronchitis virus (IBV) spike, infectious bronchitis virusmatrix, avian encephalomyelitis virus, avian reovirus, avianparamyxovirus, avian influenza virus, avian adenovirus, fowl pox virus,avian coronavirus, avian rotavirus, chick anemia virus, Salmonella spp.E coli, Pasteurella spp., Bordetella spp., Eimeria spp., Histomonas sppTrichomonas spp., Poultry nematodes, cestodes, trematodes, poultrymites/lice. and poultry protozoa.

In one embodiment of the recombinant infectious laryngotracheitis virusthe foreign DNA sequence encodes a cytokine. In another embodiment thecytokine is chicken myelomonocytic growth factor (cMGF) or chickeninterferon (cIFN). Cytokines include, but are not limited to:transforming growth factor beta, epidermal growth factor family,fibroblast growth factors, hepatocyte growth factor, insulin-like growthfactors. B-nerve growth factor, platelet-derived growth factor, vascularendothelial growth factor, interleukin 1, IL-1 receptor antagonist,interleukin 2, interleukin 3, interleukin 4, interleukin 5, interleukin6, IL-6 soluble receptor, interleukin 7, interleukin 8, interleukin 9,interleukin 10, interleukin 11, interleukin 12, interleukin 13,angiogenin, chemokines, colony stimulating factors,granulocyte-macrophage colony stimulating factors, erythropoietin,interferon, interferon gamma, leukemia inhibitory factor, oncostatin M,pleiotrophin, secretory leukocyte protease inhibitor, stem cell factor,tumor necrosis factors, and soluble TNF receptors. These cytokines arefrom humans, bovine, equine, feline, canine, porcine or avian.Recombinant ILT virus expressing cytokines is useful to enhance theimmune response when combined with vaccines containing anitgens ofdisease causing microorganisms.

Recombinant infectious laryngotracheitis virus expressing cytokines isused to enhance the immune response either alone or when combined withvaccines containing cytokines or antigen genes of disease causingmicroorganisms.

Antigenic polypeptide of a human pathogen which are derived from humanherpesvirus include, but are not limited to: hepatitis B virus andhepatitis C virus hepatitis B virus surface and core antigens, hepatitisC virus, human immunodeficiency virus, herpes simplex virus-1, herpessimplex virus-2, human cytomegalovirus, Epstein-Barr virus,Varicella-Zoster virus, human herpesvirus-6, human herpesvirus-7, humaninfluenza, measles virus. hantaan virus, pneumonia virus, rhinovirus,poliovirus, human respiratory syncytial virus. retrovirus, human T-cellleukemia virus, rabies virus, mumps virus, malaria (Plasmodiumfalciparum), Bordetellapertussis, Diptheria, Rickettsia prowazekii,Borrelia berfdorferi, Tetanus toxoid, malignant tumor antigens.

The antigenic polypeptide of an equine pathogen is derived from equineinfluenza virus, or equine herpesvirus. In one embodiment the antigenicpolypeptide is equine influenza neuraminidase or hemagglutinin. Examplesof such antigenic polypeptide are: equine influenza virus type A/Alaska91 neuraminidase and hemagglutinin, equine influenza virus type A/Prague56 neuraminidase and hemagglutinin, equine influenza virus type A/Miami63 neuraminidase, equine influenza virus type A/Kentucky 81neuraminidase and hemagglutinin, equine herpesvirus type 1 glycoproteinB, and equine herpesvirus type 1 glycoprotein D, Streptococcus equi,equine infectious anemia virus, equine encephalitis virus, equinerhinovirus and equine rotavirus.

The antigenic polypeptide of an equine pathogen is derived from bovinerespiratory syncytial virus or bovine parainfluenza virus, and iscapable of being expressed in a host infected by the recombinantinfectious bovine rhinotracheitis virus. For example, the antigenicpolypeptide is derived from bovine respiratory syncytial virusattachment protein (BRSV G), bovine respiratory syncytial virus fusionprotein (BRSV F), bovine respiratory syncytial virus nucleocapsidprotein (BRSV N), bovine parainfluenza virus type 3 fusion protein, andthe bovine parainfluenza virus type 3 hemagglutinin neuraminidase.

The foreign gene may be put under control of an endogenous upstreaminfectious laryngotracheitis virus promoter, or it may be put undercontrol of a heterologous upstream promoter. The heterologous upstreampromoter may be derived from the HCMV IE promoter, the PRV gX promoter,and BHV-1.1 VP8 promoter.

The present invention further provides a recombinant infectiouslaryngotracheitis virus comprising the infectious laryngotracheitisviral genome which contains a deletion or other alteration in the uniqueshort region of the viral genome, wherein the deletion or alteration isin the glycoprotein gG gene, so that upon replication, the recombinantvirus produces no glycoprotein gG. The following recombinant viruses arepreferred embodiments of this invention: A recombinant infectiouslaryngotracheitis virus designated S-ILT-002, S-ILT-014, S-ILT-009,S-ILT-015, and S-ILT-017.

The present invention further provides a recombinant infectiouslaryngotracheitis virus comprising the infectious laryngotracheitisviral genome which contains a deletion or other alteration in the uniqueshort region of the viral genome, wherein the deletion or alteration isin the glycoprotein gI gene, so that upon replication, the recombinantvirus produces no glycoprotein gI.

The present invention further provides a recombinant infectiouslaryngotracheitis virus comprising the infectious laryngotracheitisviral genome which contains a deletion or other alteration in the uniqueshort region of the viral genome, wherein the deletion or alteration isin the glycoprotein gG gene and in the glycoprotein gI gene, so thatupon replication, the recombinant virus produces no glycoprotein gG andno glycoprotein gI.

The present invention further provides a recombinant infectiouslaryngotracheitis virus comprising the infectious laryngotracheitisviral genome which contains a deletion in the unique short region of theviral genome, wherein the deletion is in the US2 gene, UL47-like gene,glycoprotein g60 gene. It is contemplated that a deletion in any one ofthese genes will attenuate the virus, rendering it suitable to be usedas a vaccine against infectious laryngotracheitis virus.

The present invention further provides a recombinant infectiouslaryngotracheitis virus which comprises a foreign gene inserted withinthe unique short region of the infectious laryngotracheitis viralgenome, provided. however, that the insertion is not in the proteinkinase gene, the glycoprotein gD gene, the glycoprotein gE gene and theORF10 gene. The foreign gene is inserted in such a way that it iscapable of being expressed in the recombinant infectiouslaryngotracheitis virus infected host cell. Preferred insertion sitesare the US2 gene, the UL47-like gene, the ORF4 gene and the glycoproteing60 gene.

A foreign gene may be inserted within any one of these sites in such away that it may be expressed in a host cell which is infected which therecombinant infectious laryngotracheitis virus of the present invention.

The foreign gene thus inserted may encode a screenable marker, such asE. coli β-galactosidase or E. coli β-glucuronidase.

The foreign gene thus inserted may encode an antigenic polypeptidewhich, when introduced into the host cell, induces production ofprotective antibodies against an avian disease causing agent from whichthe antigen is derived or derivable. Such antigenic polypeptide may bederived or derivable from infectious bronchitis virus, Newcastle diseasevirus, infectious bursal disease virus, and Marek's disease virus. Suchantigenic polypeptide may also be derived or derivable from avianencephalomyelitis virus, avian reovirus, avian paramyxovirus, avianinfluenza virus, avian adenovirus, fowl pox virus, avian coronavirus,avian rotavirus, chick anemia agent, Salmonella spp. E. coli,Pasterurella spp., Bordetella spp. Eimeria spp. Histomonas spp.,Trichomonas spp, Poultry nematodes, cestodes, trematodes, poultrymites/lice, poultry protozoa.

The foreign gene thus inserted may be put under control of an endogenousupstream infectious laryngotracheitis virus promoter, or it may be putunder control of a heterologous upstream promoter. The heterologousupstream promoter may be the HCMV IE promoter, the PRV gX promoter orBHV-1.1 VP8 promoter.

The present invention further provides a vaccine for infectiouslaryngotracheitis virus which comprises a suitable carrier and aneffective immunizing amount of any of the recombinant infectiouslaryngotracheitis virus of the present invention. This vaccine maycontain either inactivated or live recombinant virus.

Suitable carriers for the recombinant virus are well known in the artand include proteins, sugars, etc. One example of such a suitablecarrier is a physiologically balanced culture medium containing one ormore stabilizing agents such as hydrolyzed proteins, lactose, etc.Preferably, the live vaccine is created by taking tissue culture fluidsand adding stabilizing agents such as stabilizing, hydrolyzed proteins.Preferably, the inactivated vaccine uses tissue culture fluids directlyafter inactivation of the virus.

The present invention further provides a vaccine which comprises asuitable carrier and an effective immunizing amount of a recombinantinfectious laryngotracheitis virus comprising the infectiouslaryngotracheitis viral genome which contains a deletion in the uniqueshort region of the viral genome, wherein the deletion is in theglycoprotein gG gene. A preferred embodiment of this invention is avaccine which comprises a suitable carrier and an effective immunizingamount of any one of the following viruses: recombinant infectiouslaryngotracheitis viruses designated S-ILT-014, S-ILT-002, S-ILT-009,S-ILT-015 and S-ILT-017.

The present invention further provides a multivalent vaccine forinfectious laryngotracheitis virus and for one or more of other aviandiseases which comprises an effective immunizing amount of a recombinantvirus comprising the infectious laryngotracheitis viral genome whichcontains a deletion in the unique short region, wherein the deletion isin the glycoprotein gG gene, and an insertion of a foreign gene into anon-essential site of the viral genome.

The foreign gene encodes an antigenic polypeptide which induces hostcell production of protective antibodies against an avian diseasecausing agent from which the antigen is derived or derivable.

The foreign gene may be derived or derivable from infectious bronchitisvirus, Newcastle disease virus, infectious bursal disease virus, andMarek's disease virus, avian encephalomyelitis virus, avian reovirus,avian paramyxovirus, avian influenza virus, avian adenovirus, fowl poxvirus, avian coronavirus, avian rotavirus, chick anemia agent,Salmonella spp., E. coli, Pasteurella spp., Bordetella spp., Eimeriaspp., Histomonas spp., Trichomonas spp., poultry nematodes, cestodes,trematodes, poultry mites/lice, poultry protozoa.

The present invention further provides a vaccine which comprises asuitable carrier and an effective immunizing amount of a recombinantinfectious laryngotracheitis virus comprising the infectiouslaryngotracheitis viral genome containing a deletion or other alterationin the unique short region of the viral genome, wherein the deletion oralteration is in the glycoprotein gG gene, so that upon replication, therecombinant virus produces no glycoprotein gG. A preferred embodiment ofthis invention is a vaccine which comprises a suitable carrier and aneffective immunizing amount of any one of the following viruses:recombinant infectious laryngotracheitis viruses designated S-ILT-0 14,S-ILT-002, S-ILT-009, S-ILT-015 and S-ILT-017.

The present invention further provides a vaccine which comprises asuitable carrier and an effective immunizing amount of a recombinantinfectious laryngotracheitis virus comprising the infectiouslaryngotracheitis viral genome which contains a deletion or otheralteration in the unique short region of the viral genome, wherein thedeletion or alteration is in the glycoprotein gI gene so that uponreplication, the recombinant virus produces no glycoprotein gI.

The present invention further provides a vaccine which comprises asuitable carrier and an effective immunizing amount of a recombinantinfectious laryngotracheitis virus comprising the infectiouslaryngotracheitis viral genome which contains a deletion or otheralteration in the unique short region of the viral genome, wherein thedeletion or alteration is in the glycoprotein gG gene and theglycoprotein gI gene so that upon replication, the recombinant virusproduces no glycoprotein gG and glycoprotein gI.

The present invention further provides a vaccine which comprises asuitable carrier and an effective immunizing amount of a recombinantinfectious laryngotracheitis virus comprising the infectiouslaryngotracheitis viral genome which contains a deletion in the uniqueshort region of the viral genome, wherein the deletion is in the US2gene, UL47-like gene, or glycoprotein g60 gene.

The present invention further provides a vaccine which comprises asuitable carrier and an effective immunizing amount of a recombinantinfectious laryngotracheitis virus comprising the infectiouslaryngotracheitis viral genome which contains a deletion in the uniqueshort region of the viral genome, wherein the deletion is in the US2gene, ORF4 gene, UL47-like gene, or glycoprotein g60 gene, and insertionof a foreign gene into a non-essential site in the viral genome.

The foreign gene encodes an antigenic polypeptide which induces hostcell production of protective antibodies against an avian diseasecausing agent from which the antigen is derived or derivable.

The foreign gene may be derived or derivable from infectious bronchitisvirus, Newcastle disease virus, infectious bursal disease virus, andMarek's disease virus, avian encephalomyelitis virus, avian reovirus.avian paramyxovirus. avian influenza virus, avian adenovirus, fowl poxvirus avian coronavirus. avian rotavirus, chick anemia agent, Salmonellaspp., E coli, Pasteurella spp., Bordetella spp., Eimeria spp.,Histomonas spp., Trichomonas spp. poultry nematodes, cestodes.trematodes, poultry mites/lice, poultry protozoa.

The present invention further provides a vaccine which comprises asuitable carrier and an effective immunizing amount of a recombinantinfectious laryngotracheitis virus comprising the infectiouslaryngotracheitis viral genome which contains an insertion of a foreigngene into a non-essential site in the viral genome. The foreign geneencodes an antigenic polypeptide which induces host cell production ofprotective antibodies against an avian disease causing agent from whichthe antigen is derived or derivable.

The foreign gene may be derived or derivable from infectious bronchitisvirus, Newcastle disease virus, infectious bursal disease virus, andMarek's disease virus, avian encephalomyelitis virus, avian reovirus,avian paramyxovirus, avian influenza virus, avian adenovirus, fowl poxvirus, avian coronavirus, avian rotavirus, chick anemia agent,Salmonella spp. E. coli, Pasterurella spp., Bordetella spp. Eimeria spp.Histomonas spp., Trichomonas spp, Poultry nematodes, cestodes,trematodes, poultry mites/lice, poultry protozoa.

The present invention further provides a method of immunizing an animalagainst infectious laryngotracheitis virus which comprises administeringto chickens or other poultry an effective immunizing dose of any of thevaccines of the present invention.

The present invention further provides a method for distinguishingchickens or other poultry which are vaccinated with an effectiveimmunizing amount of a recombinant virus which produces no glycoproteingG from those which are infected with a naturally-occurring infectiouslaryngotracheitis virus. This method comprises analyzing a sample ofbody fluid from the chickens or other poultry for the presence ofglycoprotein gG of the infectious laryngotracheitis virus and at leastone other antigen normally expressed in chickens or other poultryinfected by a naturally-occurring infectious laryngotracheitis virus.The presence of antigen which is normally expressed in chickens or otherpoultry infected by a naturally-occurring infectious laryngotracheitisvirus and the absence of glycoprotein gG in the body fluid is indicativeof being vaccinated with the recombinant vaccine and not infected with anaturally-occurring infectious laryngotracheitis virus. The presence ofglycoprotein gG and the antigen in the body fluid may be determined bydetecting in the body fluid antibodies specific for the antigen andglycoprotein gG.

The present invention further provides a method for distinguishingchickens or other poultry which are vaccinated with an effectiveimmunizing amount of a recombinant infectious laryngotracheitis viruswhich produces no glycoprotein gI from those which are infected with anaturally-occurring infectious laryngotracheitis virus. This methodcomprises analyzing a sample of body fluid from the chickens or otherpoultry for the presence of glycoprotein gI of the infectiouslaryngotracheitis virus and at least one other antigen normallyexpressed in chickens or other poultry infected by a naturally-occurringinfectious laryngotracheitis virus. The presence of the antigen which isnormally expressed in chickens or other poultry infected by anaturally-occurring infectious laryngotracheitis virus and the absenceof glycoprotein gI in the body fluid is indicative of being vaccinatedwith the recombinant vaccine and not infected with a naturally-occurringinfectious laryngotracheitis virus. The presence of the antigen andglycoprotein gI in the body fluid may be determined by detecting in thebody fluid antibodies specific for the antigen and glycoprotein gI.

The present invention further provides a method for distinguishingchickens or other poultry which are vaccinated with an effectiveimmunizing amount of a recombinant virus which produces no glycoproteingG and no glycoprotein gI from those which are infected with anaturally-occurring infectious laryngotracheitis virus. This methodcomprises analyzing a sample of body fluid from the chickens or otherpoultry for the presence of glycoprotein gG and gI of the infectiouslaryngotracheitis virus and at least one other antigen normallyexpressed in an animal infected by a naturally-occurring infectiouslaryngotracheitis virus. The presence of the antigen which is normallyexpressed in chickens or other poultry by a naturally-occurringinfectious laryngotracheitis virus and the absence of glycoprotein gGand gI in the body fluid is indicative of being vaccinated with thevaccine and not infected with a naturally-occurring infectiouslaryngotracheitis virus. The presence of the antigen and glycoprotein gGand gI in the body fluid may be determined by detecting in the bodyfluid antibodies specific for the antigen and glycoprotein gG and gI.

The present invention further provides a homology vector for producing arecombinant infectious laryngotracheitis virus by inserting a foreignDNA into the unique short region of the infectious laryngotracheitisgenomic DNA, which comprises a double-stranded DNA molecule consistingessentially of a double-stranded foreign gene, which is flanked oneither side by the double-stranded DNA homologous to the DNA located inthe unique short region of the genomic DNA, provided, however, that theflanking sequences are not homologous to the glycoprotein gD gene, theglycoprotein gE gene, the protein kinase gene, and the ORF10 gene. Theforeign gene may encode a screenable marker, such as E. coliB-galactosidase or E. coli B-glucuronidase.

The present invention further provides a homology vector for producing arecombinant infectious laryngotracheitis virus by deleting DNA whichencodes a screenable marker, which has been inserted into the infectiouslaryngotracheitis virus genomic DNA, which comprises a double strandedDNA molecule consisting essentially of a double-stranded DNA to bedeleted, which is flanked on each side by a double stranded DNAhomologous to the infectious laryngotracheitis virus glycoprotein gGgene, glycoprotein gI gene, US2 gene, or UL-47 like gene. Preferredembodiments of this invention are the homology vectors designatedHomology Vector 544-55.12. Homology Vector 562-6 1. F. Homology Vector472-73.27, Homology Vector 560-52.Fl and Homology Vector 579-14.G2.

This invention provides an isolated nucleic acid molecule encoding aUSI0 gene (SEQ ID NOs:60 and 70), AvSp gene (SEQ ID NOs: 61 and 71). US2gene (SEQ ID NO:62), PK gene (SEQ ID NO:63), UL47 gene (SEQ ID NO:64),gG gene (SEQ ID NO:65), ORF5 gene (SEQ ID NO: 66). gD gene (SEQ IDNO:67), gI gene (SEQ ID NO:68), gE gene (SEQ ID NO:69). or ORF9 gene(SEQ ID NO:70).

This invention provides an isolated polypeptide encoded by the US10 gene(SEQ ID NOs:60 and 70), AvSp gene (SEQ ID NOs: 61 and 71), US2 gene (SEQID NO:62), PK gene (SEQ ID NO:63), UL47 gene (SEQ ID NO:64), gG gene(SEQ ID NO:65), ORF5 gene (SEQ ID NO: 66), gD gene (SEQ ID NO:67), gIgene (SEQ ID NO:68), gE gene (SEQ ID NO:69), or ORF9 gene (SEQ IDNO:70).

Experimental Details

Materials and Methods

Preparation of Infectious Laryngotracheitis Virus Stock Samples

Infectious laryngotracheitis virus stock samples were prepared byinfecting primary chicken embryo kidney cells (CEK; obtained fromSpafas, Inc.) or primary chicken kidney cells (CK; obtained from chickshatched from fertile eggs supplied by Hyvac) (50) in 225 cm² flasks with0.5 ml of viral stock containing 10⁵-10⁶ pfu in 1× Eagle's Basal Medium(modified) with Hank's salts (BME), 10% bromoethylamine(BEI)-treatedfetal bovine serum (FBS), 1% glutamine stock, 2%pennicillin/streptomycin (P/S) stock, and 1% sodium bicarbonate stock(these components are obtained from Irvine Scientific or an equivalentsupplier, and hereafter the growth medium is referred to as complete BMEmedium). Viral stocks were then harvested 4-5 days later. Infected mediaand cells were resuspended in complete medium containing 20% sterilewhole milk and stored frozen at −70° C.

Preparation of Infectious Laryngotracheitis Virus DNA

Four to five days after viral infection, cells and media were scrapedfrom each flask into 15 ml conical centrifuge tubes and pelleted at1700×g for 5 minutes at 4° C. Because as much as 50% of the virus may bein the media, the supernatants were saved and treated as will bedescribed below. The cell pellets were resuspended in 1 ml PBS per tube,combined and centrifuged again at 1700×g for 5 minutes. The pellets wereresuspended in 1 ml/flask of a buffer containing 10 mM Tris-HCl pH 7.5,1 mM EDTA, and 1.5 mM MgCl₂ and were incubated for 15 minutes at 4° C.Twenty five [ls of 20% NP40 per flask was added, and the mixture wasthen homogenized in a dounce homogenizer using an A pestle. Thepreparation was centrifuged at 1700×g for 10 minutes at 4° C. and thesupernatant was retained. Ten μl of 0.5 M EDTA, 50 μl of 20% SDS, and 25μl of 10 mg/ml proteinase K was added to the supernatant (per originalflask). In some cases, this was then combined with virus obtained fromthe cell media supernatants (see above). The mixture was then treated at65° C. for 1-16 hours, followed by two extractions with phenol saturatedwith 100 mM Tris-HCl, pH 8. DNA in the aqueous phase was thenprecipitated with added 3 M sodium acetate ( 1/10th volume) and 2.5 volsof 100% ethanol.

To obtain virus from the media, the cell media supernatants werecentrifuged at 23,500×g for 30 minutes, and drained well. The pellet wasresuspended in the above proteinase K-containing mixture as described.The DNA pellets were resuspended in 20 μl TE/flask and could be used atthis point for further experiments or treated further to remove RNA withpancreatic RNase A, followed by phenol extraction and ethanolprecipitation to obtain the DNA.

To prepare viral DNA minipreps, infected 10 cm. dishes were scraped intoconical centrifuge tubes and centrifuged 5 minutes at 1000×g. Cell mediasupernatants were kept and treated as above. The cell pellets were eachresuspended in 0.5 ml of 10 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.5% NP40,and incubated 10 minutes at room temperature. Ten μl of 10 mg/ml RNase Awas added, and the preparation was centrifuged 5 minutes at 1000×g.Twenty-five μl of 20% SDS and 25 μl of 10 mg/ml proteinase K was addedto the supernatant, and the entire preparation was added to the viralpellet from the cell media if it was used. The mixture was incubated at55-65° C. for one hour, extracted with buffer-saturated phenol andprecipitated by the addition of 1 ml of ethanol. The DNA pellet wasresuspended in 20 μl of TE and stored at 4° C.

Polymerase Fill-in Reaction

DNA was resuspended in buffer containing 50 mM Tris pH 7.4, 50 mM KCl, 5mM MgCl₂, and 400 micromolar each of the four deoxyribonucleotides. Tenunits of Klenow DNA polymerase (Gibco BRL) were added and the reactionwas allowed to proceed for 15 minutes at room temperature. The DNA wasphenol extracted and ethanol precipitated as above.

DNA Sequencing

Sequencing was performed using the Sequenase Kit (US Biochemicals) andα³⁵S-dATP (New England Nuclear). Reactions using both the dGTP mixes andthe dITP mixes were performed to clarify areas of compression.Alternatively, compressed areas were resolved on formamide gels.Templates were double-stranded plasmid subclones or single stranded MI13subclones, and primers were either made to the vector just outside theinsert to be sequenced, or to previously obtained sequence. Sequenceobtained was assembled and compared using Dnastar software. Manipulationand comparison of sequences obtained was performed with IBI MacVector,Superclone and Supersee Align programs from Coral Software.

Molecular Biological Techniques

Techniques for the manipulation of bacteria and DNA, including suchprocedures as digestion with restriction endonucleases, gelelectrophoresis, extraction of DNA from gels, ligation, phosphorylationwith kinase, treatment with phosphatase, growth of bacterial cultures,transformation of bacteria with DNA, and other molecular biologicalmethods are described (42, 43). The polymerase chain reaction (PCR) wasused to introduce restriction sites convenient for the manipulation ofvarious DNAs (44). In general amplified fragments were less than 500base pairs in size and critical regions of amplified fragments wereconfirmed by DNA sequencing. Except as noted, these techniques were usedwith minor variation.

Southern Blotting of DNA

The general procedure for Southern blotting was taken from Maniatis et.al. (1982) and Sambrook, et. al.(1989) (42, 43). DNA was blotted tonylon membrane (Biorad Zetaprobe) in 0.4M NaOH and prehybridized for 5minutes in a solution containing 0.25 M Na₂HPO₄, pH 7.2, 1 mM EDTA, 7%SDS at 65° C. Labeled probe was added that had been labeled by randompriming using a Genius™ non-radioactive labeling kit fromBoehringer-Mannheim. Hybridization was overnight at 65° C. Filters werewashed twice with 40 mM Na₂HPO₄, pH 7.2, 1 mM EDTA, 5% SDS and thentwice with 40 mM Na₂HPO₄, pH 7.2, 1 mM EDTA, 1% SDS for 30 minutes eachat 65° C. Detection of bound probe was performed using the BoehringerMannheim Genius™ non-radioactive detection kit.

DNA Transfection for Generating Recombinant ILT Virus

The method is based upon the CaCl₂ procedure of Chen and Okavama (1987)(45) with the following modifications. Generation of recombinant ILTvirus is dependent upon homologous recombination between ILT viral DNAand the plasmid homology vector containing the desired foreign DNAflanked by the appropriate herpesvirus cloned sequences. Plasmid DNA(10-40 mmg) was added to 250 ml of a solution having a finalconcentration of 0.25 M CaCl₂. An equal volume of a buffer containing 50mM MOPS (pH 6.95). 280 mM NaCl. and 1.5 mM Na₂HPO₄ was added to theDNA/CaCl₂ solution. After 10 minutes at room temperature, the mixturewas added dropwise to a 6 cm dish of CEK cells on maintenance media, andplaced at 39° C. for 4 to 5 hours. The cells were rinsed once with PBS,once with 20% glycerol in PBS for 2 minutes. rinsed again with PBS andfed with maintenance media. 1.5 ml of ILT viral stock was added to themedia, and the cells were incubated overnight. The next day, freshmaintenance media was added, and the cells were incubated for two moredays. The transfection stock was harvested, aliquoted, and frozen at−70° C.

Procedure for Generating ILTV Subgenomic DNA Fragments

The ability to generate herpesviruses by cotransfection of clonedoverlapping subgenomic fragments has been demonstrated for pseudorabiesvirus (46). If deletions and/or insertions are engineered directly intothe subgenomic fragments prior to the cotransfection, this procedureresults in a high frequency of viruses containing the genomicalteration, greatly reducing the amount of screening required to purifythe recombinant virus. The procedure of overlapping cosmids to maprestriction enzyme sites was employed.

A library of subclones containing overlapping ILTV subgenomic fragmentswas generated as follows. USDA ILTV Strain 83-2 has been designatedS-ILT-001. Approximately 20 μg of ILTV DNA (obtained from S-ILT-001) in0.5 ml of 10 mM Tris-HCl pH 8.0, 1 mM EDTA (TE) was sheared by passingit twice through a 25 guage needle as previously described (46). The DNAwas centrifuged through a 15-40% glycerol gradient in 50 mM Tris-HCl pH8.0. 1 mM EDTA, and 0.3 M NaCl for 5.5 hours at 274,000×g. Fractionswere analyzed on a 0.3% agarose gel, and those containing DNA of 35-50kb were pooled, diluted twofold with TE, and precipitated with one tenthvolume of 3 M sodium acetate and 2.5 volumes of ethanol. The tubes werecentrifuged for one hour at 109,000×g at 10° C. . Pellets wereresuspended, transferred to microfuge tubes, and precipitated with onetenth volume of 3 M sodium acetate and 2.5 volumes of ethanol. The DNAwas resuspended in TE. DNA ends were made blunt ended by the POLYMERASEFILL-IN REACTION. The DNA was purified by extraction with both buffersaturated phenol and ether, precipitated with sodium acetate and ethanolas above, and resuspended in TE. Half of this material was ligated with3 mg of vector, pSY1626, by the DNA ligation reaction. The vector usedwas pSY1626, which was made as follows. Cosmid pHC79 (Gibco BRL) was cutwith HindIII and 4AvaI to remove the tetracycline gene, and the endswere filled in with Klenow polymerase (FILL IN REACTION). The polylinkerfrom pWE15 (Stratagene) was ligated into this vector. The polylinker wasisolated by digestion with EcoRI, the ends were filled in with Klenowpolymerase (FILL IN REACTION), and the fragment was purified on aLMP-agarose gel. DNA ligation was performed in the presence of meltedagarose. The resulting cosmid, pSY1005, was modified at the EcoRI siteto create pSY1626 by blunt-ended insertion of a 1.5 kb HindIII-BamHIfragment from pNEO (P-L Biochemicals) containing the neomycin resistancegene. pSY1626 was cut and made blunt at the BamHI site, and ligated withsheared ILTV fragments as described above. The ligation mixture waspackaged using Gigapack XL (Stratagene) according to the manufacturersinstructions. The packaging mixture was added to AG1 cells (Stratagene)grown in the presence of maltose, and colonies were selected on LBplates containing kanamycin. Cosmid subclones containing ILTV DNA wereidentified by comparing restriction enzyme maps of individual cosmidclones to each other and to ILVTV genomic DNA to obtain a contiguoussequence of ILTV genomic DNA.

Screen for Recombinant ILTV Expressing Enzymatic MARKER Genes

When the E. coli β-galactosidase or β-glucuronidase (uidA) marker genewas incorporated into a recombinant virus the plaques containing therecombinants were visualized by a simple assay. The enzymatic substratewas incorporated (300 μg/ml) into the agarose overlay during the plaqueassay. For the lacZ marker gene the substrate Bluogal™ (halogenatedindolyl-β-D-galactosidase, Gibco BRL) was used. For the uidA marker genethe substrate X-Glucuro Chx (5-bromo-4-chloro-3-indolyl-β-D-glucuronicacid Cyclohexylammonium salt, Biosynth AG) was used. Plaques thatexpressed active marker enzyme turned blue. The blue plaques were thenpicked onto fresh cells and purified by further blue plaque isolation.In recombinant virus strategies in which the enzymatic marker gene wasremoved, the assay involves plaque purifying white plaques from abackground of parental blue plaques. Viruses were typically purifiedwith five to ten rounds of plaque purification.

Screen for Foreign Gene Expression in Recombinant ILTV Using BlackPlaque Assays

To analyze expression of foreign antigens expressed by recombinant ILTviruses, monolayers of CEK cells were infected with recombinant ILTvirus, overlaid with nutrient agarose media and incubated for 3-5 daysat 39° C. Once plaques have developed, the agarose overlay was removedfrom the dish, the monolayer rinsed once with PBS, fixed with 100%methanol for 10 minutes at room temperature and the cells air dried.After re-hydrating the plate with PBS, the primary antibody was dilutedto the appropriate dilution with PBS plus Blotto and incubated with thecell monolayer for 2 hours to overnight at room temperature. Unboundantibody was removed from the cells by washing four times with PBS atroom temperature. The appropriate secondary antibody conjugate wasdiluted 1:500 with PBS and incubated with the cells for 2 hours at roomtemperature. Unbound secondary antibody was removed by washing the cellsthree times with PBS at room temperature.The monolayer was rinsed incolor development buffer (100 mM Tris pH 9.5/100 mM NaCl/5mM MgCl2), andincubated 10 minutes to overnight at room temperature with freshlyprepared substrate solution (0.3 mg/ml nitro blue tetrazolium+0.15 mg/ml5-bromo-4-chloro-3-indolyl phosphatase in color development buffer). Thereaction was stopped by replacing the substrate solution with TE (10 mMTris, pH7.5/1 mM EDTA). Plaques expressing the correct antigen stainblack.

Purification of ILTV gG from ILT Virus or Recombinant Viruses ExpressingILTV gG

ILTV gG was purified from the media of cells infected with either wildtype ILTV or with FPV or SPV vectors expressing ILTV gG. Cells wereallowed to go to complete cytopathic effect (CPE), the media was pouredoff, and cell debris was pelleted in a table-top centrifuge. The mediawas concentrated in an Amicon concentrator using a YM30 ultrafiltrationmembrane at 15 psi. The concentrate was dialyzed against 20 mM Tris-HCl,pH 7.0 and loaded onto a DEAE-Sephacel (Pharmacia) column equilibratedwith the same buffer. The material was eluted using a salt gradient from0 to 1.5 M NaCl in 20 mM Tris-HCl, pH 7.0. Three ml fractions werecollected and assayed by Western blot. A peptide antibody against ILTVgG was used to identify fractions containing ILTV gG. Fractions werepooled and further concentrated in a Centricon-10 microconcentrator(Amicon).

Growth of Chicken Kidney Cells and ILT Virus

An ILTV virus, designated fowl laryngotracheitis challenge virus, lotnumber 83-2, was obtained from the National Veterinary ServicesLaboratories, USDA/APHIS, Ames, Iowa. ILTV viruses were grown in primarychicken kidney cells (CK) obtained by dissection of kidneys from 6-9 dayold SPF chicks, obtained from Hy-Vac Laboratory Eggs Co. Fresh kidneycells were minced and disassociated with 5 mg/ml trypsin and were thenpelleted and resuspended at 1.3×10⁶ cells/ml. Growth media (GM) was 1×Eagle's Basal Medium (modified) with Hank's salts, with added 10% binaryethyleneimine-treated fetal bovine serum (FBS), 2 mM glutamine, 200units/ml penicillin, 200 mg/ml streptomycin, and 8.9 mM sodiumbicarbonate (85). After resuspension, cells were plated and incubated at39° C. Cells were rinsed and fed after 24 hours with maintenance media(MM), which is GM with 1% FBS. CKs were inoculated with ILTV at 0.01 to0.1 MOI and viral stocks were harvested 4-5 days later by scraping andsonicating. Titers were typically 10⁵-10⁶ μ/ml.

Preparation of Viral DNA

Cells and media from infected flasks were pelleted at 1700 g for 5′ at4° C. Supernatant and cell pellet were initially treated separately.Virion particles were centrifuged out of the supernatant at 23,500 g for30 minutes. The original cell pellet was rinsed with PBS and spun again.This pellet was resuspended in 1 ml/flask of a buffer containing 10 mMTris-HCl pH 7.5, 1 mM EDTA, and 1.5 mM MgCl₂ and incubated 15′, 4° C. Tothis was added 25 μl/flask of 20% NP40, and the mixture was douncehomogenized using an A pestle. The preparation was centrifuged at 1700g, 10′, 4° C., and the supernatant was retained and the pelletdiscarded. To the supernatant was added (per original flask) 10 μl of0.5 M EDTA, 50 μI of 20% SDS, and 25 μl of 10 mg/ml proteinase K. Thismixture was used to resuspend the pellet of viral particles obtained byhigh speed centrifugation of the first supernatant. The mixture wastreated at 65° C. for 1-16 hours, extracted twice with buffer-saturatedphenol, and precipitated with added salt and ethanol. The resulting DNApellet was resuspended in 100 μl TE/flask. This was treated further toremove RNA with pancreatic RNase A, followed by phenol extraction andethanol precipitation to obtain the DNA.

Creation of the Cosmid Library

The cosmid library of ILTV DNA was created following the protocol of vanZijl et al., (83). Approximately 20 μg of ILTV DNA in 0.5 ml of 10 mMTris-HCl, pH 8.0, 1 mM EDTA (TE) was sheared by passing it twice througha 25 gauge needle. The DNA was centrifuged through a 15-40% glycerolgradient in 50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.3 M NaCl for 5.5 h at274,000 g. Fractions were analyzed on a 0.3% agarose gel, and thosecontaining DNA of 35-50 kb were pooled, diluted twofold with TE, andprecipitated with added salt and ethanol. The tubes were spun 1 h at 10°C. and 109,000 g. Pellets were resuspended and reprecipitated with addedsalt and ethanol. The DNA was resuspended in TE and the ends were madeblunt by treatment with T4 DNA polymerase for 2 h at 15° C., in thepresence of appropriate buffer and 25 μM dNTP, followed by treatmentwith Klenow polymerase for 16 h at 15° C. using 0.25 mM dNTP. The DNAwas extracted with phenol and then ether, precipitated with added saltand ethanol, and resuspended in TE. This material was ligated overnightwith 3 μg of cosmid vector pSY1626. Cosmid pSY1626 was made by digestingcosmid pHC79 (BRL) with HindIII and AvaI to remove the tetracyclinegene. The remaining fragment and the EcoRI digested polylinker frompWE15 (Stratagene) were filled in with Klenow polymerase and ligatedtogether. The resulting cosmid vector, pSY1005, was modified at theEcoRI site to create pSY1626 by blunt-ended insertion of a 1.5 kbHindIII-BamHI fragment from pNEO (P-L Biochemicals) containing thekanamycin resistance gene. PSY1626 was cut and made blunt at the BamHIsite for use as the cosmid vector. The ligation mixture was packagedusing Gigapack XL (Stratagene) according to the manufacturer'sdirections. Colonies were selected on LB plates containing kanamycin.

Sequencing

Manual sequencing was performed using ³⁵S-dATP (NEN) with the BRLSequenase Kit which uses the dideoxyribonucleotide chain terminationmethod described by Sanger et al. (80). Reactions using both dGTP anddITP mixes were performed to clarify areas of compression.Alternatively, compressed areas were resolved on 8% acrylamide gels thatwere 40% in formamide. Automatic fluorescence sequencing was performedusing an Applied Biosystems (ABI) 373A DNA Sequencer. Subdlones weremade to facilitate sequencing. Internal primers were synthesized on anABI 392 DNA synthesizer. Sequence was obtained for both strands and wasassembled using DNAstar software. Manipulation and comparison ofsequences was performed with DNAstar programs, Superclone and Superseeprograms from Coral Software. Comparisons with GenBank were performed atthe NCBI using the BLAST network service (58).

Homology Vector 501-94

The plasmid 501-94 was constructed for the purpose of deleting a portionof the thymidine kinase (TK) gene coding region from the ILT virus (28).It incorporates the HCMW IE promoter and a screenable marker, the E.coli lacZ gene, flanked by ILT virus DNA. The HCMV IE promoter-E. coillacZ gene is inserted in the opposite transcriptional orientation to theILTV TK gene. Upstream of the marker gene is an approximately 1087 basepair fragment of ILTV DNA which includes the first 77 amino acid codonsof the ILTV TK gene. Downstream of the lacZ gene is an approximately 675base pair fragment of ILTV DNA which includes 80 amino acid codons atthe 3′ end of the ILTV TK gene. When this plasmid is used according tothe DNA TRANSFECTION FOR GENERATING RECOMBINANT ILT VIRUS, it willreplace the DNA coding for amino acids 78 to 285 of the ILTV TK genewith DNA coding for the lacZ gene. The lacZ marker gene is under thecontrol of the human cytomegalovirus (HCMV) immediate early (IE) genepromoter and also contains the pseudorabies virus (PRV) gX genepolyadenylation signal at the 3′ end of the gene. A detailed descriptionof the plasmid is given in FIGS. 5A-5D. It was constructed from theindicated DNA sources utilizing standard recombinant DNA techniques (42,43). The plasmid vector is derived from an approximately 3002 base pairHindIII fragment of pSP64/65 (Promega). Fragment 1 is an approximately1087 base pair HindIII to BclI subfragment of the ILTV 2.4 kb HindIIIfragment. Fragment 2 is an approximately 5017 base pair SalI to SalIfragment containing the HCMV IE promoter, β-galactosidase (lacZ) markergene, and PRV gX polyadenylation signal (see FIGS. 5A-5D). Fragment 3 isan approximately 675 base pair Bcl to HindIII subfragment of the ILTV2.4 kb HindIII fragment.

Homology Vector 544-55.12

The plasmid 544-55.12 was constructed for the purpose of deleting aportion of the US2 gene coding region from the ILT virus and inserting aforeign DNA. It incorporates a screenable marker, the E. coli uidA geneflanked by ILT virus DNA. The PRV gX promoter-E. coliuidA gene isinserted in the opposite transcriptional orientation to the ILTV US2gene. Upstream of the uidA gene is an approximately 2300 base pairfragment of ILTV DNA which includes 41 amino acid codons at the 3′ endof the US2 gene (SEQ ID NO 2: aa. 188-229). Downstream of the uidA geneis an approximately 809 base pair fragment of ILTV DNA which includes 22amino acid codons at the 5′ end of the US2 gene (SEQ ID NO 2: aa. 1-22).When this plasmid is used according to the DNA TRANSFECTION FORGENERATING RECOMBINANT ILT VIRUS, it will replace the ILTV US2 DNAcoding for amino acids 23 to 187 with DNA coding for the E. coli uidAgene. The uidA marker gene is under the control of the pseudorabiesvirus (PRV) gX promoter and also contains the herpes simplex virus type1 thymidine kinase (HSV-1 TK) gene polyadenylation signal at the 3′ endof the gene. A detailed description of the plasmid is given in FIGS.6A-6D. It was constructed from the indicated DNA sources utilizingstandard recombinant DNA techniques (42, 43). The plasmid vector isderived from an approximately 2958 base pair Asp718I restrictionfragment of a pSP18/pSP19 fusion such that the multiple cloning site isEcoRI/SacI/Asp718l/SacI/EcoRI. Fragment 1 is an approximately 2300 basepair Asp718I to DraI subfragment (SEQ ID NO 1: Nucl. 1-405) of the ILTV2.5 kb Asp718I fragment. Fragment 2 is an approximately 3039 base pairXbaI fragment containing the PRV gX promoter, the E. coli uidA gene, andthe HSV-1 TK polyadenylation site (See FIGS. 6A-6D). Fragment 3 is anapproximately 809 base pair XbaI to Asp718I subfragment of the ILTV 1097bp Asp718I fragment (SEQ ID NO 1: Nucl. 905-1714).

Homology Vector 562-61.1F

The plasmid 562-61.1F was constructed for the purpose of deleting partof the gI gene from the ILT virus and inserting a foreign DNA. Itincorporates a screenable marker, the E. coli uidA gene, flanked by ILTvirus DNA. The PRV gX promoter-E. coli uidA gene is transcribed in theopposite direction to the ILTV gI gene promoter. The 983 base pairdeletion begins 12 base pairs upstream of the translation initiationcodon and deletes 324 of 363 amino acid codons at the 5′ end of the ILTVgI gene (SEQ ID NO 11: aa. 325-363). When this plasmid is used accordingto the DNA TRANSFECTION FOR GENERATING RECOMBINANT ILT VIRUS, it willreplace the DNA coding for the ILTV gI gene with DNA coding for the E.coli uidA gene. A detailed description of the plasmid is given in FIGS.7A-7D. It was constructed from the indicated DNA sources utilizingstandard recombinant DNA techniques (42, 43). The plasmid vector isderived from an approximately 2647 base pair Asp718I to HindIII fragmentof pUC19. Fragment 1 is an approximately 1619 base pair Asp718I to XbaIsubfragment of the ILTV 8.0 kb Asp718I fragment (SEQ ID NO 1: Nucl.7556-9175). Fragment 2 is an approximately 691 base pair XbaI to XhoIfragment (SEQ ID NO 1: Nucl. 9175-9861) generated by the polymerasechain reaction (PCR). The template was the ILTV 8.0 kb Asp718I fragment.The upstream primer 92.09 (5′-CCTAGCACCCTTGTATCGCG-3′; SEQ ID NO. 55)sits down at a site 821 base pairs upstream of the ILTV gI gene andsynthesizes DNA toward the 3′ end of the gene. The downstream primer92.11 (5′-CGCCTCGAGTCCCAATGAATAGGCATTGG-3′; SEQ ID NO. 56) sits down ata site 12 base pairs upstream of the translation start site of the ILTVgI gene and synthesizes DNA toward the 5′ end of the gD gene. Theproduct of the PCR reaction is 818 base pairs. This DNA fragment isdigested with XbaI at the 5′ end (a restriction enzyme site present inthe ILTV DNA) and XhoI at the 3′ end (a restriction enzyme site createdin the PCR primer-see underlined sequence) to create an approximately691 base pair XbaI to XhoI fragment. Fragment 3 is an approximately 3051base pair SalI fragment containing the PRV gX promoter, the uidA gene,and the HSV-1 TK polyadenylation site (See FIGS. 6A-6D). Fragment 4 isan approximately 624 base pair XhoI to HindIII fragment generated by PCR(SEQ ID NO 1: Nucl. 10,847-11,461). The template was the ILTV 8.0 kbAsp718I fragment. The upstream primer 92.10(5′-CGCCTCGAGGACCCATGGTTGCGTGCG-3′; SEQ ID NO. 57) sits down at a site117 base pairs upstream from the translation termination codon withinthe ILTV gI gene. The downstream primer 92.08(5′-CTCGTCCGAACGAGTTACAG-3′; SEQ ID NO.58) sits down at a site 604 basepairs downstream of the translation termination site of the ILTV gI geneand within the ILTV gE gene. The PCR product (729 base pairs) isdigested with XhoI which is a unique site generated by the upstream PCRprimer (underlined) and with HindIII at a site within the ILTV gE gene.Restriction endonuclease digestion with XhoI and HindIII creates anapproximately 624 base pair Fragment 4. Fragment 5 is an approximately2700 base pair HindIII subfragment of the ILTV 8.0 kb Asp718I fragment(SEQ ID NO 1: Nucl. 11,461-13,473 plus unsequenced DNA).

Homology Vector 472-73.27.

The plasmid 472-73.27 was constructed for the purpose of deleting aportion of the glycoprotein G (gG) gene coding region from the ILT virusand inserting a foreign DNA. It incorporates a screenable marker, the E.coli lacZ gene, flanked by ILT virus DNA. The HCMV IE promoter-E. colilacZ gene is transcribed in the same direction to the ILTV gG genepromoter. The 874 base pair deletion of the ILTV gG gene extends from 60nucleotides upstream of the translation initiation site to 814nucleotides into the amino acid coding sequence, removing the codingcapacity of 271 of 292 amino acids of the gG protein (SEQ ID NO 7). Whenthis plasmid is used according to the DNA TRANSFECTION FOR GENERATINGRECOMBINANT ILT VIRUS, it will replace the DNA coding for amino acids 1to 271 of the ILTV gG gene with DNA coding for the E. coli lacZ gene. Adetailed description of the plasmid is given in FIGS. 4A-4D. It wasconstructed from the indicated DNA sources utilizing standardrecombinant DNA techniques (42, 43). The plasmid vector is derived froman approximately 2686 base pair Asp718I restriction fragment of pUC 19(Gibco, BRL). Fragment 1 is an approximately 2830 base pair Asp718I toNheI subfragment of the ILTV 5164 bp Asp718I fragment (SEQ ID NO 1:Nucl. 1714-4544). Fragment 2 is an approximately 5017 base pair SalI toSalI fragment containing the HCMV IE promoter, E. coli β-galactosidase(lacZ) marker gene, and PRV gX polyadenylation signal (see FIGS. 4A-4D).Fragment 3 is an approximately 1709 base pair SalI to Asp718Isubfragment of the ILTV 5164 bp Asp718I fragment (SEQ ID NO 1: Nucl.5419-6878).

Homology Vector 560-52.F1

The plasmid 560-52.F1 was constructed for the purpose of deleting partof the UL47-like gene all of ORF4, and part of the ILTV gG gene from theILT virus and inserting a foreign DNA. It incorporates a screenablemarker, the E. coli uidA gene. flanked by ILT virus DNA. The PRV gXpromoter-E. coli uidA gene is transcribed in the opposite direction tothe ILTV UL47-like, ORF4, and gG gene promoters. The 2640 base pairdeletion removes 442 of 511 amino acid codons at the 3′ end of theUL47-like gene (SEQ ID NO 4), the entire coding sequence of the ORF4gene (SEQ ID NO 5) and 271 of 293 amino acid codons at the 5′ end of theILTV gG gene (SEQ ID NO 7). When this plasmid is used according to theDNA TRANSFECTION FOR GENERATING RECOMBINANT ILT VIRUS, it will replacethe DNA coding for the ILTV UL47-like, ORF4 and gG genes with DNA codingfor the PRV gX promoter-E. coli uidA gene. A detailed description of theplasmid is given in FIGS. 8A-8D. It was constructed from the indicatedDNA sources utilizing standard recombinant DNA techniques (42, 43). Theplasmid vector is derived from an approximately 2958 base pair Asp718Irestriction fragment of pSP18/pSP19 such that the multiple cloning siteis EcoRI/SacI/Asp718I/SacI/EcoRI. Fragment 1 is an approximately 1066base pair Asp718I to BssHII subfragment of the ILTV 5164 bp Asp718Ifragment (SEQ ID NO 1: Nucl. 1714-2777). Fragment 2 is an approximately123 base pair SalI to BclI subfragment of the ILTV 5164 bp Asp718Ifragment. Fragment 3 is an approximately 3027 base pair BamHI fragmentcontaining the PRV gX promoter, the uidA gene, and the HSV-1 TKpolyadenylation site (See FIGS. 8A-8D). Fragment 4 is an approximately1334 base pair Bc/I to Asp718I subfragment of the ILTV 5164 bp Asp718Ifragment (SEQ ID NO 1: Nucl. 5544-6878).

Homology Vector 579-14.G2

The plasmid 579-14.G2 was constructed for the purpose of deleting theentire gG gene and a portion of the g60 gene from the ILT virus andinserting a foreign DNA. It incorporates a PRV gX promoter and ascreenable marker, the E. coli uidA gene, flanked by ILT virus DNA. ThePRV gX promoter-E. coli uidA gene is transcribed in the same directionto the ILTV gG and g60 gene promoters. The 3351 base pair deletionincludes the entire coding sequence of the ILTV gG gene (SEQ ID NO 7)and 733 of 986 amino acid codons from the 5′ end of the g60 gene (SEQ IDNO 8). When this plasmid is used according to the DNA TRANSFECTION FORGENERATING RECOMBINANT ILT VIRUS, it will replace the DNA coding for theILTV gG gene and amino acids 1 to 733 of the ILTV g60 gene with DNAcoding for the E. coli uidA gene. A detailed description of the plasmidis given in FIGS. 9A-9D. It was constructed from the indicated DNAsources utilizing standard recombinant DNA techniques (42, 43). Theplasmid vector pUC19 (Gibco, BRL) is derived from an approximately 2677base pair Asp718I to BamHI fragment. Fragment 1 is an approximately 2830base pair Asp718I to NheI subfragment of the ILTV 5164 bp Asp718Ifragment (SEQ ID NO 1: Nucl. 1714-4544). Fragment 2 is an approximately3051 base pair SalI fragment containing the PRV gX promoter, E. coliβ-glucuronidase (uidA) marker gene, and an HSV-1 TK polyadenylation site(See FIGS. 9A-9D). Fragment 3 is an approximately 1709 base pair SalI toBamHI subfragment of the ILTV 4545 base pair BamHI fragment (SEQ ID NO1: Nucl. 7895-9604).

Plasmid 544-39.13

Plasmid 544-39.13 contains the β-glucuronidase expression cassetteconsisting of the PRV gX promoter, E. coli β-glucuronidase (uidA) markergene, and an HSV-1 TK polyadenylation site. A detailed description ofthe marker gene is given in FIGS. 10A-10D. It was constructed utilizingstandard recombinant DNA techniques (42, 43) by joining restrictionfragments from the following sources with the synthetic DNA sequencesindicated in FIGS. 10A-10D. The plasmid vector pSP71 (Promega) isderived from an approximately 3066 base pair XmaI to SmaI fragment.Fragment 1 is an approximately 422 base pair SalI to EcoRI restrictionsubfragment of the PRV BamHI restriction fragment #10 (47). Note thatthe EcoRI site was introduced at the location indicated in FIGS. 12A-12Dby PCR cloning. Fragment 2 is an approximately 1826 base pair EcoRI toSmaI fragment of the plasmid pRAJ260 (Clonetech). Note that the EcoRIand XmaI sites were introduced at the locations indicated in FIGS.10A-10D by PCR cloning. Fragment 3 is an approximately 784 base pairXmaI subfragment of the HSV-1 BamHI restriction fragment Q (48). Notethat this fragment is oriented such that the polyadenylation sequence(AATAAA) is located closest to the junction with the E. coli uidA gene.

Plasmid 388-65.2

Plasmid 388-65.2 contains the β-galactosidase expression cassetteconsisting of the HCMV immediate early (IE) promoter, the E. coli lacZmarker gene, and the PRV gX gene polyadenylation site. A detaileddescription of the β-galactosidase expression cassette is given in FIGS.11A-11D. It was constructed utilizing standard recombinant DNAtechniques (42, 43) by joining restriction fragments from the followingsources with the synthetic DNA sequences indicated in FIGS. 11A-11D. Theplasmid vector pSP72 (Promega) is derived from an approximately 3076base pair PstI to PstI fragment. Fragment 1 is a 1154 base pair PstI toAvaII fragment derived from a HCMV 2.1 kb PstI fragment containing theHCMV IE promoter. Fragment 2 is a 3010 base pair BamHI to PvuII fragmentderived from plasmid pJF751 (49) containing the E. coli lacZ gene.Fragment 3 is an approximately 750 base All pair NdeI to SalI fragmentderived from PRV BamHI #7 which contains the carboxy-terminal 19 aminoacids and the polyadenylation signal of the PRV gX gene.

EXAMPLES Example 1

Complete Sequence of the Unique Short Region of InfectiousLaryngotracheitis Virus (ILTV)

The sequence of 13,473 base pairs of contiguous DNA from the shortregion of the ILT virus (SEQ. ID. NO. 1) was determined. This sequencecontains the entire 13,098 base pair unique short region as well as 273base pairs of repeat region at one end and 102 base pairs of repeatregion at the other end. The unique short region contains 13 methionineinitiated open reading frames (ORF) of greater than or equal to 110amino acids (excluding smaller nested ORFs). All 13 ORFs were aligned tothe Entrez release 6.0 virus division of the Genbank DNA databaseutilizing the IBI MacVector Protein to DNA alignment option (defaultsettings). Eight of the ORFs exhibited significant homology to one ormore other virus genes (see Table I). The nucleotide sequence numbersreferred to below begin within the internal repeat sequence and endwithin the terminal repeat sequence. The unique short region begins atbase pair 274 of SEQUENCE ID NO. 1.

TABLE I Sequence Homology between Infectious Laryngotracheitis Virus(ILTV) Open Reading Frames in the Unique Short Region and other ViralProteins Open Reading Genbank Frame (ORF) Start (BP) End (BP) Length(aa) Allignmenta 1 (Rc)^(b) 970 281 229 EHV-1 US2 2 1059 2489 476 MDV PK3 2575 4107 510 HSV-1 UL47 4 4113 4445 110 NSc 4 (RC) 4519 4139 126 NS 54609 5487 292 PRV gX 6 5697 8654 985 ILTV g60 6 (RC) 7826 6948 292 HSV-2UL39 7 8462 9766 434 PRV g50 8 9874 10962 362 VZV gI 8 (RC) 11150 10617177 NS 9 11159 12658 499 VZV gE 10 12665 13447 260 NS ^(a)Sequenceallignment scored to the Entrez Release 6.0 of Genbank Virus Database.^(b)RC = Reverse Complement. ^(c)NS = No score above 120 was found.Other Abbreviations: EHV = Equine herpesvirus; MDV = Mareks diseasevirus; HSV-1 = Herpes Simplex virus 1; PRV = Pseudorabies virus; ILTV =Infectious laryngotracheitis virus; HSV-2 = Herpes Simplex virus 2; VZV= Varicella-Zoster virus; BP = base pairs; aa = amino acids.US2 gene

The US2 gene consists of 690 base pairs and codes for a protein 229amino acids in length and molecular weight approximately 25,272 daltons(SEQ. ID. NO. 12, 13). The ILTV US2 is homologous to the Equineherpesvirus(EHV)-1 and EHV-4 US2 proteins. The US2 gene is transcribedfrom nucleotide 970 to 281 on the reverse complement strand of the ILTVunique short region (SEQ. ID. NO. 1). The function of the US2 geneproduct is unknown.

Protein Kinase Gene

The protein kinase gene consists of 1431 base pairs from nucleotide 1059to 2489 and codes for a protein 476 amino acids in length and molecularweight approximately 54,316 daltons (SEQ. ID. NO. 2). The ILTV proteinkinase is homologous to the protein kinases from Mareks disease virus(MDV) Equine herpesvirus(EHV)-1 and -4, Pseudorabies virus (PRV),Varicella-Zoster virus (VZV), Simian varicella virus (SVV), and HerpesSimplex virus(HSV)-1 and -2.

UL47-Like Gene

The UL47-like gene is unique in its location within the unique shortregion of ILT virus. The UL47-like gene in all other known herpesvirusesis located within the unique long sequence. The UL47-like gene consistsof 1533 base pairs from nucleotide 2575 to 4107 and codes for a protein510 amino acids in length and molecular weight approximately 57,615daltons (SEQ. ID. NO. 3).

ORF4

ORF4 codes for a protein of unknown function. ORF4 consists of 333 basepairs from nucleotide 4113 to 4445 and codes for an open reading frame110 amino acids in length and molecular weight approximately 12,015daltons (SEQ. ID. NO. 4).

ORF4 Reverse Complement

ORF4 Reverse Complement (RC) codes for a protein of unknown function.ORF4 RC consists of 380 base pairs from nucleotide 4519 to 4139 andcodes for an open reading frame 126 amino acids in length and molecularweight approximately 13,860 daltons (SEQ. ID. NOS. 14, 15).

gG Gene

The gG gene consists of 879 base pairs from nucleotide 4609 to 5487 andcodes for a glycoprotein 292 amino acids in length and molecular weightapproximately 31,699 daltons (SEQ. ID. NO. 5). ILTV gG glycoprotein ishomologous to PRV gX, Bovine herpesvirus(BHV)-1.3 gG, EHV-1 gG and EHV-4gG. Recombinant ILTV gG protein produced in a Swinepox virus vector or aFowlpox virus vector can be purified (see Materials and Methods) andreacts to peptide antisera to ILTV gG. The peptide antisera reacts toILTV gG from wild type virus, but not to viruses deleted for the ILTV gGgene. Deletion of the gG gene results in an attenuated ILT virus that isuseful as a vaccine against ILT disease in chickens (see table inExample 6) and also serves as a negative marker to distinguishvaccinated from infected animals.

g60 Gene

The g60 gene has been identified as glycoprotein 60 (33, 53). The g60gene consists of 2958 base pairs from nucleotide 5697 to 8654 and codesfor a glycoprotein 985 amino acids in length and molecular weightapproximately 106,505 daltons (SEQ. ID. NO. 6).

ORF6 Reverse Complement

ORF6 RC consists of 878 base pairs from nucleotide 7826 to 6948 andcodes for an open reading frame 292 amino acids in length and molecularweight approximately 32,120 daltons (SEQ. ID. NO. 16, 17). The ILTV ORF6RC shares limited homology to portions of the HSV-1 and HSV-2ribonucleotide reductase large subunit (UL39).

gD Gene

The expression of the gD glycoprotein in vectored Fowlpox virus orherpesvirus of turkeys (33) is sufficient to raise a protective immuneresponse in the chicken. The gD gene consists of 1305 base pairs fromnucleotide 8462 to 9766 and codes for a glycoprotein 434 amino acids inlength and molecular weight approximately 48,477 daltons (SEQ. ID. NO.10, 11). The ILTV gD glycoprotein is homologous to the PRV g50, and thegD from HSV-1. MDV. IPV, and BHV-1.1. Monoclonal antibodies raised toILT virus react specifically with gD protein from ILTV and also react toILTV gD protein expressed in a Herpesvirus of Turkeys (HVT) virusvector. ILTV gD expressed in the HVT vector is useful as a subunitvaccine.

gI Gene

The gI gene consists of 1089 base pairs from nucleotide 9874 to 10,962and codes for a glycoprotein 362 amino acids in length and molecularweight approximately 39,753 daltons (SEQ. ID. NO. 7). The ILTV gIglycoprotein is homologous to the VZV gI. Recombinant ILTV gI proteinexpressed in a swinepox virus vector reacts to convalescent sera fromILTV-infected chickens. Deletion of the gI gene results in an attenuatedILT virus that is useful as a vaccine against ILT disease in chickens.Recombinant viruses deleted for gI are safe in animal trials whenvaccinated by a natural route directly into the respiratory tract,whereas parental virus causes lesions in 90% of the birds inoculated viathe same route. Deletion of the gI gene serves as a negative marker todistinguish vaccinated from infected animals.

ORF8 Reverse Complement

ORF8 Reverse Complement codes for a protein of unknown function. ORF8 RCconsists of 533 base pairs from nucleotide 11,150 to 10,617 and codesfor an open reading frame 177 amino acids in length and molecular weightapproximately 19,470 daltons (SEQ. ID. NO. 18, 19).

gE Gene

The gE gene consists of 1500 base pairs from nucleotide 11,159 to 12.658and codes for a glycoprotein 499 amino acids in length and molecularweight approximately 55,397 daltons (SEQ. ID. NO. 8). The ILTV gEglycoprotein is homologous to the gE glycoproteins from VZV, Simianherpesvirus (SHV), EHV-1, HSV-1, and PRV. The ILTV gE is a neutralizingantigen useful as a subunit vaccine.

ORF10

ORF10 consists of 783 base pairs from nucleotide 12,665 to 13,447 andcodes for a protein 261 amino acids in length and molecular weightapproximately 27,898 daltons (SEQ. ID. NO. 9).

Example 2

S-ILT-004

S-ILT-004 is an infectious laryngotracheitis virus (ILTV) that has anapproximately 620 base pair deletion of the thymidine kinase (TK) gene(28). The gene for E. coli β-galactosidase (lacZ) was inserted in theplace of the TK gene and is under the control of the HCMV immediateearly (IE) promoter. Transcription of the HCMV IE promoter-lacZ gene isin the opposite orientation to the TK promoter.

S-ILT-004 was constructed using homology vector 501-94 (see Materialsand Methods) and S-ILT-001 (USDA ILTV Strain 83-2) in the DNATRANSFECTION FOR GENERATING RECOMBINANT ILT VIRUS. The transfectionstock was screened by the Bluogal™ SCREEN FOR RECOMBINANT HERPESVIRUSEXPRESSING ENZYMATIC MARKER GENES. The result of blue plaquepurification was recombinant virus S-ILT-004. This virus wascharacterized by restriction mapping and the SOUTHERN BLOTTING OF DNAprocedure. This analysis confirmed the presence of the β-galactosidase(lacZ) marker gene and the deletion of approximately 619 base pairs ofthe TK gene. The remaining TK gene sequence codes for protein includingamino acids 1 to 77, and amino acids 286 to 363. The HCMV IEpromoter-lacZ gene is in the opposite orientation to the TK genetranscription.

S-ILT-004 is attenuated by deletion of the ILTV TK gene, but retainsother genes known to be involved in the immune response in chickens toILT virus. Therefore, S-ILT-004 may be useful as a killed vaccine toprotect chickens from ILT disease.

Example 3

S-ILT-009

S-ILT-009 is an infectious laryngotracheitis virus (ILTV) that has anapproximately 498 base pair deletion of the ILTV US2 gene and anapproximately 874 base pair deletion of the ILTV gG gene. The gene forE. coli β-glucuronidase (uidA) was inserted in the place of the US2 geneand is under the control of the pseudorabies virus (PRV) gX promoter.

S-ILT-009 was constructed using homology vector 544-55.12 (see Materialsand Methods) and S-ILT-002 in the DNA TRANSFECTION FOR GENERATINGRECOMBINANT ILT VIRUS. S-ILT-002 was constructed as described in Example5 (S-ILT-014). The transfection stock was screened by the X-Gluc SCREENFOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES. Theresulting purification of a blue plaque was recombinant virus S-ILT-009.This virus was characterized by restriction mapping and the SOUTHERNBLOTTING OF DNA procedure. This analysis confirmed the presence of thePRV gX promoter-β-glucuronidase (uidA) marker gene and the deletion ofapproximately 498 base pairs of the ILTV US2 gene and an approximately874 base pair deletion of the ILTV gG gene. However, during the Bluogal™SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES, adeletion of the HCMV IE promoter-lacZ gene was detected within theexisting ILTV gG deletion. The remaining insert into the ILTV gGdeletion contains approximately 2000 base pairs of DNA of which all ofthe lacZ gene and part of the,PRV gX polyadenylation site are missing.The deletion was characterized by detailed restriction mapping anddetermined to be slightly different from the S-ILT-014 deletion (SeeExample 5).

S-ILT-009 is attenuated by deletion of the ILTV US2 and gG genes, butretains other genes known to be involved in the immune response inchickens to ILT virus. Therefore, S-ILT-009 is useful as an attenuatedlive vaccine or as a killed vaccine to protect chickens from ILT diseaseas shown in the table. Since S-ILT-009 does not express the ILTV gGgenes, it is utilized as a negative marker to distinguish vaccinatedanimals from infected animals as described previously.

TABLE II EFFICACY OF RECOMBINANT LIVE ILT VIRUS S-ILT-009 AGAINSTVIRULENT INFECTIOUS LARYNGOTRACHEITIS VIRUS CHALLENGE Gene(s) VaccineDeleted Dose Route Challenge^(a) Protection^(b) S-ILT-009 gG-, US2-  7.8× 10³ IO^(c) OS^(d) 70% S-ILT-009 gG-, US2- 1.56 × 10³ IO OS 77%Controls OS  0% ASL IO OS 90% embryo 14 day old chicks ^(a)USDAChallenge virus = 1.0 × 10^(4.5) pfu ^(b)Protection = # healthybirds/total (%). ^(c)Intraocular ^(d)Orbital Sinus

Example 4

S-ILT-011

S-ILT-011 is an infectious laryngotracheitis virus (ILTV) that has anapproximately 983 base pair deletion of the ILTV gI gene. The gene forE. coli β-glucuronidase (uidA) was inserted in the place of the gI geneand is under the control of the pseudorabies virus (PRV) gX promoter.The PRV gX promoter-uidA gene is in the opposite orientation to thedirection of transcription of the ILTV gI promoter.

S-ILT-011 was constructed using homology vector 562-61.1F (see Materialsand Methods) and S-ILT-001 in the DNA TRANSFECTION FOR GENERATINGRECOMBINANT ILT VIRUS. The transfection stock was screened by the X-GlucSCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES.The result of blue plaque purification was recombinant virus S-ILT-011.This virus was characterized by restriction mapping and the SOUTHERNBLOTTING OF DNA procedure. This analysis confirmed the presence of theβ-glucuronidase (uidA) marker gene and the deletion of approximately 983base pairs of the ILTV gI gene which deletes 325 of 363 amino acidcodons from the 5′ end of the gI gene.

S-ILT-011 is attenuated and is useful as a killed vaccine to protectchickens from ILT disease. S-ILT-011 shows a small plaque phenotype intissue culture which is indicative of slow viral growth and attenuation.Since S-ILT-0 11does not express the ILTV gI gene, it may be utilized asa negative marker to distinguish vaccinated animals from infectedanimals. As indicated in Example 1, ILTV-infected chickens makeantibodies against ILTV gI protein.

Example 5

S-ILT-013

S-ILT-013 is an infectious laryngotracheitis virus (ILTV) that has anapproximately 983 base pair deletion of the ILTV gI gene and anapproximately 874 base pair deletion of the ILTV gG gene (and a deletionof the HCMV IE promoter lacZ marker gene making the lacZ genenonfunctional). The gene for E. coli β-glucuronidase (uidA) was insertedin the place of the gI gene and is under the control of the pseudorabiesvirus (PRV) gX promoter.

S-ILT-013 was constructed using homology vector 562-61.1F (see Materialsand Methods) and S-ILT-014 in the DNA TRANSFECTION FOR GENERATINGRECOMBINANT ILT VIRUS. The transfection stock was screened by the X-GlucSCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES.The result of blue plaque purification was recombinant virus S-ILT-013.This virus was characterized by restriction mapping and the SOUTHERNBLOTTING OF DNA procedure. This analysis confirmed the presence of theβ-glucuronidase (uidA) marker gene and the deletion of approximately 983base pairs of the ILTV gI gene which removes 325 of 363 amino acidcodons from the 5′ end of the gI gene. This analysis also confirmed anapproximately 874 base pair deletion of the ILTV gG gene and anapproximately 1906 base pair insertion of a partial HCMV IEpromoter-lacZ marker gene DNA, of which a portion of the HCMV IEpromoter and almost none of the lacZ gene remains (see Example 6).

S-ILT-013 is attenuated and is useful as a killed vaccine to protectchickens from ILT disease. S-ILT-013 shows a small plaque phenotype intissue culture which is indicative of slow viral growth and attenuation.Since S-ILT-013 does not express the ILTV gI or gG genes, ILTV gI and gGmay be utilized as negative markers to distinguish vaccinated animalsfrom infected animals.

Example 6

S-ILT-014

S-ILT-014 is an infectious laryngotracheitis virus (ILTV) that has anapproximately 874 base pair deletion of the ILTV gG gene and a deletionof the inserted HCMV IE promoter lacZ marker gene making the lacZ genenonfunctional. S-ILT-014 was derived from a purified S-ILT-002 virusstock in which a deletion of the HCMV IE promoter lacZ marker geneoccurred.

S-ILT-002 was constructed using homology vector 472-73.27 (See Materialsand Methods) and S-ILT-001 in the DNA TRANSFECTION FOR GENERATINGRECOMBINANT ILT VIRUS. The virus S-ILT-002 has a 874 base pair deletionwithin the ILTV gG gene and an insertion of the E. coli β-galactosidase(lacZ) gene in place of the ILTV gG gene. However, during the Bluogal™SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES, awhite plaque was picked which contained a deletion of the lacZ genewithin the ILTV gG deletion.

This virus, S-ILT-014, was characterized by restriction mapping, DNASEQUENCING and the SOUTHERN BLOTTING OF DNA procedure. This analysisconfirmed the presence of an approximately 874 base pair deletion of theILTV gG gene and approximately 1956 base pair insertion of a partialHCMV IE promoter lacZ marker gene DNA (2958 base pairs deleted). Theremaining HCMV IE promoter lacZ marker gene DNA consists of anapproximately 686 base pair DNA fragment of the approximately 1154 basepair HCMV IE promoter and an approximately 1270 base pair DNA fragmentcontaining approximately 520 base pairs of the 3010 base pairβ-galactosidase (lacZ) marker gene and all of the approximately 750 basepair PRV gX polyadenylation signal.

S-ILT-014 is useful as an attenuated live vaccine or as a killed vaccineto protect chickens from ILT disease as indicated in the table below.Since S-ILT-014 does not express the ILTV gG gene and ILTV-infectedchickens make antibodies to gG as indicated in Example 1. ILTV gG isutilized as a negative marker to distinguish vaccinated animals frominfected animals.

TABLE III EFFICACY OF RECOMBINANT LIVE ILT VIRUS S-ILT-014 AGAINSTVIRULENT INFECTIOUS LARYNGOTRACHEITIS VIRUS CHALLENGE Gene(s) VaccineDeleted Dose Route Challenge^(a) Protection^(b) S-ILT-014 gG- 1.08 × 10⁴IO^(c) OS^(d) 97% S-ILT-014 gG- 2.16 × 10³ IO OS 97% Controls OS  0% ASLembryo IO OS 90% 14 day old chicks ^(a)USDA Challenge virus = 1.0 ×10^(4.5) pfu ^(b)Protection = # healthy birds/total (%). ^(c)Intraocular^(d)Orbitual Sinus

Example 7

S-ILT-015

S-ILT-015 is an infectious laryngotracheitis virus (ILTV) that has anapproximately 2640 base pair deletion of the UL47-like gene. the ORF4gene. and ILTV gG gene. The gene for E. coli β-glucuronidase (uidA) wasinserted in the place of the UL47-like, ORF4, and gG genes and is underthe control of the pseudorabies virus (PRV) gX promoter. The PRV gXpromoter-uidA gene is in the opposite orientation to the direction oftranscription of the ILTV UL47-like, ORF4, and gG promoters.

S-ILT-015 was constructed using homology vector 560-52.F1 (see Materialsand Methods) and S-ILT-001 in the DNA TRANSFECTION FOR GENERATINGRECOMBINANT ILT VIRUS. The transfection stock was screened by the X-GlucSCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES.The result of blue plaque purification was recombinant virus S-ILT-015.This virus was characterized by restriction mapping and the SOUTHERNBLOTTING OF DNA procedure. These results confirmed the presence of a2640 base pair deletion which includes 442 of a total 511 amino acidcodons at the 3′ end of the UL47-like gene, all of the ORF4 gene and 271of 293 amino acid codons of the 5′ end of the gG gene.

S-ILT-015 is useful as an attenuated live vaccine or as a killed vaccineto protect chickens from ILT disease as indicated in the table below.Since S-ILT-015 does not express the ILTV gG gene, ILTV gG is utilizedas a negative marker to distinguish vaccinated animals from infectedanimals.

TABLE IV EFFICACY OF RECOMBINANT LIVE ILT VIRUS S-ILT-015 AGAINSTVIRULENT INFECTIOUS LARYNGOTRACHEITIS VIRUS CHALLENGE Gene(s) VaccineDeleted Dose Route Challenge^(a) Protection^(b) S-ILT-015 gG-, 1.0 × 10⁵IO^(c) OS^(d) 70% UL47-like Controls OS  0% ASL IO OS 90% embryo 14 dayold chicks ^(a)USDA Challenge virus = 1.0 × 10^(4.5) pfu ^(b)Protection= # healthy birds/total (%). ^(c)Intraocular ^(d)Orbital Sinus

Example 8

S-ILT-017

S-ILT-017 is an infectious laryngotracheitis virus (ILTV) that has anapproximately 3351 base pair deletion of the ILTV gG gene, ORF4 gene andthe g60 gene. The gene for E. coli β-glucuronidase (uidA) was insertedin the place of the ILTV gG and g60 genes and is under the control ofthe pseudorabies virus (PRV) gX promoter.

S-ILT-017 was constructed using homology vector 579-14.G2 (see Materialsand Methods) and S-ILT-001 in the DNA TRANSFECTION FOR GENERATINGRECOMBINANT ILT VIRUS. The transfection stock was screened by the X-GlucSCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES.The result of blue plaque purification was recombinant virus S-ILT-017.

S-ILT-017 is attenuated by deletion of the ILTV g60 and gG genes. butretains other genes known to be involved in the immune response inchickens to ILT virus. Therefore, S-ILT-017 may be used as a killedvaccine to protect chickens from ILT disease. Since S-ILT-017 does notexpress the ILTV gG or g60 genes, it is used as a negative marker todistinguish vaccinated animals from infected animals.

Example 9

Recombinant Infectious Laryngotracheitis Viruses that Express InfectiousBronchitis Virus (IBV) Spike and Matrix Protein Genes:

A homology vector is used to generate ILT viruses containing the IBVArkansas spike protein gene. The recombinant ILT virus contains adeletion of one or more ILTV genes, including gG, US2, UL47-like, andORF4, and the insertion of two foreign genes: the E. coliβ-glucuronidase gene (uidA) and the IBV Arkansas spike protein gene. TheuidA gene is under the control of the PRV gX promoter and the IBVArkansas spike protein gene is under the control of the HCMV IEpromoter.

To construct a homology vector containing the foreign genes insertedinto the ILT virus, a DNA fragment containing the HCMV-IE promoter, theIBV Arkansas spike protein and the HSV-1 TK polyadenylation signal isinserted into a restriction enzyme site at the position of the deletionof the ILTV gG gene in the ILTV homology vector. A DNA fragmentcontaining the PRV gX promoter and the E. coli β-glucuronidase (uidA)gene is inserted into a unique restriction enzyme site within the ILTVhomology vector. A recombinant virus is constructed by combining thefinal homology vector containing the IBV Arkansas spike gene and the E.coli β-glucuronidase (uidA) gene and S-ILT-001 in the DNA TRANSFECTIONFOR GENERATING RECOMBINANT ILT VIRUS. The transfection stock is screenedby the X-Gluc SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATICMARKER GENES to detect the presence of the uidA gene and by the BLACKPLAQUE ASSAY FOR FOREIGN GENE EXPRESSION to detect the presence of theIBV Arkansas spike protein.

A similar strategy is used to construct recombinant ILT viruses carryingthe IBV SI protein from Arkansas, Massachusetts,or Connecticutserotypes, IBV matrix protein from Arkansas, Massachusetts, orConnecticut serotypes, and IBV nucleocapsid from Arkansas, Mass. orConnecticut serotypes. The strategy is also used to constructrecombinant ILT viruses carrying the Newcastle Disease virus (NDV) HNand F genes and the Infectious Bursal Disease virus (IBDV) polyproteinor portions thereof. The stratey is also used to construct recombinantILT viruses carrying the Mareks Disease virus (MDV) gA, gD, and gBgenes.

Recombinant ILT virus carrying these antigens are valuable as amultivalent vaccine to protect chickens from diseases caused by ILTV andone or more of the viruses IBV, NDV, IBDV, or MDV. Since the ILTVvaccines described here do not express ILTV gG, it is useful as anegative marker to distinguish vaccinated animals from infected animals.

Example 10

Vaccines Utilizing ILTV to Express Antigens from Various Disease CausingMicroorganisms

Antigens from the following microorganisms are utilized to developpoultry vaccines: Chick anemia agent, Avian encephalomyelitis virus,Avian reovirus, Avian paramyxoviruses, Avian influenza virus ,Avianadenovirus. Fowl pox virus, Avian coronavirus, Avian rotavirus,Salmonella spp., E coli., Pasteurella spp., Haemophilus spp., Chlamydiaspp., Mycoplasma spp., Campylobacter spp., Bordetella spp., Poultrynematodes, cestodes, trematodes, Poultry mites/lice, Poultry protozoa(Eimeria spp., Histomonas spp., Trichomonas spp.).

Example 11

A Genomic Map of Infectious Laryngotracheitis Virus and the Sequence andOrganization of Genes Present in the Unique Short Region

A cosmid library of the ILTV genome was created to facilitaterestriction endonuclease mapping. Forty-three overlapping cosmids wereanalyzed by digestion with Asp718I and NotI, Asp718I was known to cutthe genome relatively infrequently (63), and it was found that NotI cutthe genome less than ten times, which enabled cutting the vector awayfrom the ILTV DNA insert. Comparison of these cosmid digests allowed theorder of the Asp718I fragments covering 85% of the ILTV genome to bedetermined (FIG. 12). On the long end of the genome, seven cosmids wereidentified which all contained a NotI site 0.9 kb from the end of thecloned insert; all other cosmid inserts had heterogeneous ends fromshearing. This 0.9 kb fragment was used as a probe (P1 in FIG. 12) togenomic ILTV digested with Asp718I, NotI, or BamHI, the sizes of thegenomic fragments that hybridized were identical to the size of thefragments excised from the cloned cosmid insert, indicating that thecloned insert extended all the way or very close to the end of theunique long. The 0.9 kb fragment did not hybridize to other bands in theILTV digest, consistent with previous reports that this virus resemblesPRV, and contains no long repeat (66). Once the cosmid clones wereordered, the restriction sites for a more frequent cutting enzyme,BamHI, were mapped.

The resulting map indicated that the cosmid library did not includeclones from the unique short portion of the genome. Cosmids spanning theunique short region of HVT (76) and PRV (83) have been found to beunderrepresented in cosmid libraries. The Asp718I fragments found in thecosmid clones with an Asp718I digest of wild type ILTV and identifiedfragments of 8.0, 5.1, and 2.5 kb which were not represented in thecosmid library (FIG. 13) were compared. These fragments were cloned intoplasmids and hybridized to each other and to ILTV digested with BamHI.The Asp718I 2.5 and 8.0 kb fragments cross-hybridized, indicating thatthey contained sequence repeated in both clones. Fine mapping of theAsp718I 2.5 and 8.0 kb fragments showed them to contain 2.1 kb ofidentical sequence. Hybridization to ILTV digested with BamHI identifiedBamHI bands of 7.5, 6.5, and 4.5 kb which overlapped the Asp718Ifragments. These BamHI fragments were cloned and analyzed by restrictiondigestion and hybridization. This allowed the map of the entire uniqueshort region and some of the flanking short repeat to be elucidated(FIG. 13). Subclones of this region were made, and the entire uniqueshort region was sequenced.

To complete the genomic map, the map searched for an Asp718I or BamHIfragment that spanned the region between the short repeat sequences ofthe 8.0 or 2.5 kb Asp718I fragments mentioned above and the unique longregion identified in the cosmid map. A 10 kb NotI fragment from therightmost end of cosmid D5 (FIG. 12) was hybridized to genomic ILTVdigests on Southern blots. Interestingly, ladders of hybridizing bandswere seen when the enzymes BamHI, NotI, and Asp718I were used. The bandscorresponding to these ladders were not generally visible in ethidiumbromide stained gels. Subsequent subcloning and mapping of the 10 kb D5fragment indicated that it contained up to 5 repeats of an 856 bpsegment, and that the cosmid insert ended within a repeat motif.HinduIII, which cuts once within the repeat, was used to clone the 856bp fragment. When this fragment (FIG. 12, P2) was used to probe ILTVdigested with SfiI, NotI, Asp718I, and BamHI, ladders of hybridizationwere again seen (FIG. 14). These ladders arise from varying numbers ofthe 856 bp repeat in different viral molecules. SfiI cuts only once inthis ILTV strain, and a ladder at very high molecular weight can beseen. Because the unique short is expected to invert, two overlappingSfiI ladders containing the unique short and terminal repeat (TR_(s))should be present.; however, the bands are too large in this region tomake this distinction. NotI and Asp718I cut further away from therepeat, generating ladders beginning at 10.5 or 12 kb. The Asp718Idigest should generate two overlapping ladders, because one fragment isbounded by an Asp718I site in the unique long, while the other isbounded by the end of the TR_(s). In contrast, only one ladder should begenerated by the NotI digest. Comparison of FIG. 14 lane c (NotI) withlane d (Asp718l) does suggest that in lane d a second ladder issuperimposed on the first, starting somewhat higher. BamHI cuts close tothe repeated region, and a ladder beginning at 3.4 kb is found. HindIIIcuts within the repeat and generates a strongly hybridizing 856 bp band,as well as the two flanking HindIII fragments of about 1.1 and 2.5 kb,which each contain a portion of the repeated sequence. The presence ofthis 856 bp repeat accounted for the occasional observation of very finesubmolar bands in ethidium bromide-stained Asp718I digests. It alsoaccounted for the lack, in ethidium bromide-stained gels, of a molar orhalf-molar quantity Asp718I or BamHI band greater than 10 kb, which wasexpected to span this region based on analysis of the cosmid clones.Instead, because of the presence of the 856 bp repeat, this band existsas many submolar bands comprising the ladder. As can be seen in theBanHI digest, there can be thirteen or more repeats of the region.Comparison of the repeat sequence to the sequence submitted to GenBankby Johnson et al. (67) indicated that it corresponded (99% identity) tonucleotides 1140 to 1996 of their sequence, which is a region justupstream of the ILTV ICP4 gene. The relationship of the repeat to thesurrounding sequence is depicted in FIG. 15. Restriction digestsindicate that the region to the right of the repeat as shown is similarin the two strains; however, the position of the BamHI site indicated tothe left of the repeat differs between them.

To identify the remainder of the short repeat from the 856 bp repetitiveregion to the BamHI fragments used for sequencing the unique short, the8.0 kb Asp718I fragment containing part of the short repeat was used asa probe to a second cosmid library of ILTV. One cosmid, clone 2F12,hybridized to the probe. Restriction endonuclease analysis of 2F12 andcomparison to the cosmid map indicated that it was not a singlecontiguous cosmid, but was composed of two large non-contiguousfragments (see FIG. 12). The break in the rightmost fragment was withina repeat of the 856 bp region. This fragment included at least two 856bp repeats, and extended 4.6 kb through the remainder of the shortrepeat into the unique short.

To identify the end of the TR_(s), the 6.6 kb NotI fragment spanning theunique long and the short internal repeat (IR_(s)) (P3 in FIG. 2) wasused as a probe. It was noted that a 2.9 kb NotI fragment seen in gelsstained with ethidium bromide was not represented in the restrictionendonuclease map, and considered that it might represent the end of theTR_(s). Hybridization of a NotI digest of ILTV with P3 indicated thatthis was indeed the case (FIG. 16). The 2.9 kb NotI band hybridizes, asdoes the 6.6 kb band corresponding to the probe. In the BamHb digest,the predicted 13 kb fragment containing a portion of the IR_(s) and a3.5 kb fragment corresponding to the end of the TR_(s) are evident. Inthe Asp718I digest, an overlapping 2.7 kb fragment from the unique longhybridizes, and the high molecular weight ladder described previouslywas seen.

Sequencing of the ILTV unique short and flanking region identified nineopen reading frames in the unique region and two (duplicated) in therepeat region as diagrammed in FIG. 13 (SEQ UD NO:59). Comparison of theproteins encoded by these ORFs to the GenBank database (BLAST homologysearch, National Center for Biological Information NCBI) demonstratedidentity for most of the potential proteins with other known herpesvirusgene products. Table V summarizes the closest homologies found for eachgene and gives the probability scores for those homologies as generatedby the search program. ORF2 (SEQ ID NO:63), the protein kinase (PK) gene(SEQ ID NO:63), is the most highly conserved of the ILTV ORFs to itsherpes homologues. In contrast, the glycoprotein genes are lessconserved. It should be noted that portions of the sequences of the ILTVprotein kinase, gG, and ORF 5 genes have been published (69, 70 and 81);however, these genes were mapped to the unique long region. Adescription of each of the nine unique short genes and the two genes inthe flanking short repeat follows.

The first open reading frame in the unique short encodes a 229 aaprotein showing identity to other herpesvirus US2 proteins (SEQ IDNO:62). Like other US2 genes, it is in the opposite orientation to theremaining ORFs in the unique short. The coding sequence of the gene endsjust within the unique short region, and a potential poly-A additionsite is found 115 bases downstream in the short repeat. Two possibleTATA promoters are found 37 and 70 bases upstream from the initiationcodon.

ORF2 encodes a protein kinase with strong identity to many otherherpesvirus protein kinases and to cellular protein kinases. Theorganization of the US2 and PK genes, with their 5′ ends close togetherand their promoters possibly overlapping, is similar to that found inother herpesviruses. Two TATA sequences are present 14 and 49 basesupstream of the PK start codon, and two polyadenylation signals arefound, one immediately after the stop codon, and one 50 basesdownstream.

ORF3 encodes a 623 aa protein with similarity to the herpes simplexvirus UL47 gene (SEQ ID NO:64). The program comparing this protein withother UL47 proteins projects a poor probability score for this homology.However, at least one of the regions of identity between ILTV and HSVUL47 corresponds to a region that is conserved among other herpesvirusUL47 homologues, suggesting that this identity is significant (FIG. 17).Additionally, it should be noted that equally poor probability scoresfor homology generated by comparisons of the gG or gI genes are alsoseen for certain homologue pairings, suggesting that these scores arenot sufficient for determining homology. It is interesting that the ILTVUL47 gene, normally found in the unique long region of otherherpesviruses, appears to have been transposed into the unique short inILTV.

The fourth open reading frame encodes a 292 aa glycoprotein homologousto PRV gG (SEQ ID NO:65). Four N-linked glycosylation sites with theconsensus sequence NXT or NXS are present. The protein has a signalsequence of 26 aa, which could be cleaved at G/AP, but lacks atransmembrane anchor. It is therefore likely that this protein issecreted, similar to other herpesvirus gG homologues. This gene has aconsensus TATA sequence 83 bases upstream from the ATG start, and hastwo potential polyadenylation sites 73 and 166 bases downstream from thestop codon.

ORF5 could encode a protein of 985 amino acids (SEQ ID NO:66). Ahydrophobic signal sequence is found at the amino terminus. and ahydrophobic sequence is present at the carboxy terminus. Nineglycosylation sites are found, suggesting that this is a glycoprotein.ORF 5 contains an imperfect repeat. consisting of 30 to 36 bp repeatedapproximately 23 times from amino acid 431 to amino acid 677. Thehydrophilic amino acid consensus sequence created by this repeat isFTQTPSTEPET/A. Comparison of ORF 5 with other herpesvirus sequences(Table V) shows similarity to the glycoprotein product from the equineherpesvirus 1 US5 gene (EUS5, 82). The low probability score for thisidentity arises primarily from the fact that both genes containthreonine-rich repeats. It is not clear whether this reflects homologyin form, function, or both. Both the EUS5 and the ILTV ORF 5 genes arelarge, have similar positions among flanking genes in the unique short,have signal sequences, and encode glycoproteins, but other sequencesimilarities are not seen. It is interesting that the ORF 5 repeatregion shows similarity to mucin genes, which also contain threoninerich repeats. The human mucin gene, for example, has the repeatGTQTPTTTPITTTTTVTPTPTPT, where 7 of the first 11 amino acids areidentical to the ORF 5 repeat sequence. Again, whether this reflects asimilarity in function of the encoded proteins is unclear. A TATAsequence is found 560 bases upstream of the start codon; the nearestconsensus polyadenylation signal is at the end of the gI gene. Thissuggests that the ORF 5 transcript may be coterminal with the gDtranscript.

The open reading frame for the gD homologue (ORF 6) (SEQ ID NO:67)overlaps the end of ORF 5. Four in-frame methionines are found withinthe first 58 amino acids of the open reading frame, and it is not clearwhich is the actual translational start codon. Because a potential TATApromoter sequence is located only 6-9 bases upstream from the firstpossible ATG codon, this codon would probably not be within RNAtranscribed from this promoter; however, there are several TATAsequences further upstream that may also be used to initiatetranscription. The other three potential initiation codons are found ataa 23, 47, and 58 within this ORF. Comparison of the sequencessurrounding the four ATGs with the eukaryotic translational initiationconsensus sequence A/GCCATGG (71) suggests that the latter two ATGcodons max, be preferred translational start sites. The proteinsequences derived from each of these starts were examined for thepresence of eukaryotic signal sequences and signal cleavage sites. Astart at aa 58 within the ORF would result in a signal peptide of 26amino acids with a predicted cleavage site between two alanine residues.This same signal sequence would be positioned much further from theamino terminus and embedded in a more hydrophilic sequence if the otherstart sites were used. The start of ILTV gD was tentatively assigned toposition 58, which would result in a protein 377 amino acids long. Ofcourse, it is possible that more than one initiation codon is used invivo. Experiments of Zelnik et al. (88) suggest that alternate in-frameATG codons are used to initiate MDV and HVT gD transcription in vitro,though the in vivo situation was not addressed. Additional experimentson gD transcription and translation in ILTV are necessary to identifyits translational start codon.

The ILTV gD homologue has a secretory signal sequence and atransmembrane helix (aa 352-372) at the carboxy terminus. Only onepotential glycosylation site is found at position 250-252; this is ofthe form NPS, and may not be glycosylated due to the proline residue.There is some question, therefore, as to whether processed ILTV gDcontains N-linked oligosaccharides. This would be similar to the gDhomologue in pseudorabies virus, gp50, which also lacks N-linkedglycosylation sites (75). As in other herpesviruses, the gD codingsequence lacks a poly-A addition signal immediately following the gene,and the closest signal is at the end of the gI gene.

The seventh open reading frame encodes a protein of 362 aa and is mosthomologous to varicella zoster virus glycoprotein I (SEQ ID NO:68). Theencoded protein shows all the characteristics of related gIglycoproteins, including a signal sequence with a potential cleavagesite at positions 22 and 23 between a glycine and an isoleucine, atransmembrane helix at the carboxy terminus from 272-292, and fourpossible N-linked glycosylation sites. A TATA sequence is present 51bases upstream from the methionine start codon.

Two possible poly-A addition signals are found within the codingsequence for ILTV gI, and may be the signals used by the gD and ORF 5transcription units upstream.

The gE gene (ORF 8) follows the gI. This gene is 499 aa long. andcontains four N-linked glycosylation sites (SEQ ID NO:69). A signalsequence of 18 amino acids is present, and there are two and possiblythree membrane-associated helices in the carboxy terminal portion of theprotein. The gE gene has a TATA box 86 bases upstream of the startcodon, and a potential poly-A addition signal just prior to the 3′ endof the coding region. This may serve as the polyadenylation site for thegI gene.

The ninth open reading frame extends across the junction of the uniqueshort and the short repeat, and could encode a protein of 260 aminoacids (SEQ ID NO:70). This protein has no signal sequence or membraneanchor, but has one possible N-linked glycosylation site. In a search ofGenBank, some similarity is found between this protein and BLRF2 of EBV,but the significance of this similarity is unknown. The poly-A additionsignal in the short repeat may be utilized by this gene. A potentialTATA sequence is found 178 bases upstream of the first ATG of this ORF.

The first open reading frame in the short repeat (SRORF1) (SEQ ID NOs:61 and 71) encodes a 294 aa protein which displays homology to the geneproduct of MDV SORF3 (79 and 84) and HVT ORF3 (87). In MDV and HVT, thecorresponding gene is found as one copy in the unique short, and itsfunction is unknown. No homology has been identified with mammalianherpesviruses; this gene appears to be specific to avian herpesviruses.MDV SORF3 has been deleted by Parcells et al. (74), and does not appearto be absolutely required for infection in chickens.

SRORF2 encodes a protein of 278 amino acids with homology to otherherpesvirus US10 genes (SEQ ID NOs:60 and 72). A zinc finger motif,found in the EHV-4 US10, is highly conserved in the ILTV US10 (aminoacids 201-218); this suggests that the ILTV US10 gene is a DNA bindingprotein. Regulatory sequences include a poly-A addition signal 163 bpafter the stop codon; it is unclear where the promoter for this generesides.

Discussion

The organization of the genes in the unique short region of ILTV issimilar to that seen in other herpesviruses. Several genes encodingglycoproteins are present, and the order of these genes is similar tothat seen in equine herpesvirus 1, particularly with respect to ORF 5.Similarities to avian herpesviruses are also evident in the presence ofthe avian-specific gene. SRORF1, and its position relative to US2 andPK, though it differs from HVT and MDV in that it is in the short repeatand is duplicated, also appearing downstream from the ORF 9 gene. The PKgene itself has the most identity to MDV and HVT PK genes; however,other genes are found to be more like their homologues in diverseherpesviruses such as EHV, PRV, and SHV SA8. Unusual characteristics ofthe ILTV unique short are the inclusion of a gene normally found in theunique long, the UL47 homologue, and the presence of the unique gene,ORF 5, which contains a set of degenerate repeats.

This analysis of the structure of ILTV disagrees with previous reports.Comparison of the sequences described here with those of the AustralianILTV isolate SA-2 indicates that a 32 kd protein described by Kongsuwanet al. (70) is almost identical to the gG in this application, and thesequenced fragment of the g60 protein presented by Kongsuwan et al. (69)is part of the ORF 5 gene in this application. However, they identifiedthe 5 kb Asp718I fragment containing both of these genes as coming fromthe unique long region of SA-2 (66). Recently, Guo et al. (62) reportedthe sequence of a region from the USDA challenge strain which theyascribed to the unique short on the basis of comparison to the mappresented by Johnson et al. (66). No identity was found between thissequence and the unique short sequence described here. Instead, thesequence described by Guo et al. (62) shows 98% identity to a sequencerecently submitted to GenBank by Johnson et al. (67 and 68), which isreported to encode the ICP4 gene of ILTV. The BamHI sites within theICP4 coding region generate two contiguous fragments of 1.2 and 1.7 kb(see FIG. 15). In the map described here, two contiguous BamHI fragmentsof this size are found within the short repeats (FIG. 12). In addition,the 856 bp repeat element, which is found just upstream of the ICP4 gene(FIG. 15), was mapped in this application within the short repeats. Thisindicates that the ICP4 gene in the strain used in these studies ispresent in the IR_(s) and the TR_(s). It is possible, but unlikely, thatthe Australian SA-2 vaccine strain underwent an unusual rearrangementwhich altered the relationship of the unique long, unique short, andshort repeat. However, Guo et al. (62) used the same challenge strain asthe one described in this application, and the sequence they reported isnot in the unique short, but in the short repeats, similar to the ICP4genes of other herpesviruses.

The gene encoded by ORF 5 contains threonine rich, degenerate repeats.These are similar in composition and in their repetitive nature torepeats found in mucin genes. This repeated region in mucin is modifiedby O-linked oligosaccharides and is highly hydrophilic. It isinteresting to speculate on what the function of this somewhat similarregion might be in infection, if it is expressed in toto in ILTV. Atleast a portion of this gene is known to be expressed, as Kongsuwan etal. (69) cloned and sequenced a fragment from it by probing a lambdagt11 library with a monoclonal antibody that was known to bind to a 60kd ILTV protein (g60) on Western blots (86). The relationship of such a60 kd protein to the predicted 985 aa product from ORF 5 is unknown.Comparison of the application sequence with the complete sequence of theg60 coding region (81) shows a 98.5% homology between the SA-2 strainand the USDA strain. Interestingly, there is an insertion of a block of10 amino acids in g60 relative to the ORF 5 protein; this differencereflects one additional degenerate repeat sequence in the SA-2 strain.

As mentioned above, Kongsuwan et al. (70) described an ILT′ gene thatencoded a 32 kd protein with similarity to PRV gG. A comparison of theILTV gG protein sequence described in this application with their 32 kdprotein found 10 amino acid differences in the first 273 residues of theprotein. At amino acid 274, a deletion of one base pair in SA-2 relativeto the USDA strain created a frame shift, such that 19 additionalresidues were found in the challenge strain as opposed to 26 in SA-2. Apeptide was made from the carboxy terminal sequence elicited antisera inmice which reacted with ILTV gG; this indicates that the sequencedescribed in this application reflects the actual carboxy terminus inthe USDA strain. A similar situation was found when the ILTV gD proteindescribed in this application was compared with the ILTV gD sequencesubmitted to GenBank by Johnson et al. (68). Ten differences were foundin the first 419 amino acids, after which a deletion of a base in theSA-2 strain relative to the the sequence described in this applicationcaused the predicted carboxy termini to differ, with 15 more amino acidsin the USDA strain and 9 in SA-2. These differences could arise fromerrors introduced during cloning and sequencing of these genes. It isalso possible that the carboxy termini of the ILTV gG and gD genes arevariable between these strains.

The 856 bp repeat unit identified within the short repeat is justupstream of the ICP4 gene described by Johnson et al. (67), but, fromthe sequence alone, it does not appear to be repetitive in the SA-2strain. The BamHI fragment containing this repetitive region is 2848 bplong in SA-2. The smallest repeat, seen faintly in the BamHI ladder ofFIG. 14, is 3.4 kb long. This is not quite large enough to include tworepeats, and suggests that other alterations between the two strains mayexist in this region. A repeat of this sort has not been previouslydescribed for this or other ILTV strains, though the submolar nature ofthe bands may have obscured its presence. The appearance of the ladderis reminiscent of defective interfering particles, but it is notbelieved that this represents a case of defective interfering particlesin the viral stock used here. Several reasons for this follow. 1)Defective interfering particles are generally found when viruses arepassaged at high multiplicity, and the ILTV viral stocks of thisapplication were passaged at low multiplicity. In fact, viral stocksoriginating from a single picked plaque exhibited similar ladders whentheir DNA was subjected to Southern blot analysis, suggesting that asingle viral particle containing a set number of repeats couldregenerate the full range of the ladder after being grown for a shortperiod of time. 2) If populations of defective interfering particleswere present, one might expect to encounter digest fragments that wouldnot be accommodated in the linear viral map (see. for example, 77), yetall but one of the cosmids analyzed make a contiguous map, with Asp718Ibands identical to those present in genomic ILTV digests. The exception,2F12, was unusual in being the only one of several hundred cosmid clonesscreened which contained part of the unique short. This probablyrepresented an aberrant cloning event, and not a widespread phenomenonrelated to defective viral particles. 3) Defective interfering particlesoften are present in larger molar amounts than standard viral particles,such that restriction fragments originating from the defective particlesare overrepresented. In contrast, the bands of the 856 bp ladder aresubmolar, and are only rarely visible in ethidium bromide stained gels.4) Defective interfering particles contain origins of replication. The856 bp repeat itself does not contain a herpesvirus origin ofreplication as defined by the consensus sequence of Baumann et al. (59).From these considerations it was concluded that varying numbers of 856bp units are present in the short repeats of standard viral DNA from theUSDA challenge strain of ILTV. Since fragments exist that containthirteen or more repeats of the region, genomic DNA from ILTV could varyby over 11 kb in the short repeat regions. Repetitive regions have beenidentified in other herpesviruses; for example, Marek's disease viruscontains a 132 bp repetitive sequence in the long repeat regions (61 and73) and expansion of this repeat is associated with reduction of viraloncogenicity. The presence of the 856 bp tandem repeats in ILTV, incontrast, does not appear to affect viral pathogenicity, since thisstrain does cause severe clinical disease in chickens. It would beinteresting to examine other ILTV strains for the presence of thisrepeat.

Table V indicates the ORFs of the ILTV unique short and the HSVnomenclature for these genes, in those cases where homology is found.The third column shows the best matches from the Blast homology search(NCBI), and the probability scores assigned by the program for thematches indicated. Smaller numbers indicate less likelihood that thematch could occur randomly.

A genomic map of infectious laryngotracheitis virus (ILTV) and a 18.912bp sequence containing the entire unique short region and a portion ofthe flanking short repeats is presented. In determining the genomic map,an 856 bp region repeated as many as 13 times was identified within theshort repeats. The unique short sequence contains 9 potential openreading frames (ORFs). Six of these ORFs show homology to other knownherpesvirus unique short genes. Using the herpes simplex virusnomenclature, these genes are the US2, protein kinase, and glycoproteinsG, D, I, and E (SORFs 1, 2, 4, 6, 7, and 8, respectively).Interestingly, an open reading frame with homology to HSV-1 UL47 (SORF3)is found in the unique short. One very large open reading frame (ORF 5 )is present and contains a threonine rich, degenerate repeat sequence.This gene appears to be unique to ILTV among sequenced herpesviruses.Two ORFs were identified within the short repeat region. SRORF1 ishomologous to a gene (SORF3) found in the unique short region in bothMDV and HVT, and appears to be specific to avian herpesviruses. SRORF2has homology to HSV US10.

TABLE V HSV ORF Homolog Best Matches Blast Score 1 US2 EHV1 EUS1 3.1 ×10⁻¹³ EHV4 EUS1 5.3 × 10⁻¹² HSV2 US2 6.7 × 10⁻⁷ 2 PK MDV PK 8.2 × 10⁻³⁶HVT PK 5.4 × 10⁻³⁵ HSV1 PK 4.1 × 10⁻³⁰ 3 UL47 HSV1 UL47 6.0 × 10⁻¹ EHV1UL47 9.9 × 10⁻¹ MDV UL47 9.9 × 10⁻¹ 4 gG PRV gG 5.3 × 10⁻⁵ BHV1 gG 1.7 ×10⁻² EHV1 gG 6.8 × 10⁻¹ 5 ORF 5 EHV1 EUS5 1.9 × 10⁻⁴⁵ Human mucin 1.1 ×10⁻²⁵ 6 gD MDV gD 6.8 × 10⁻⁴ PRV g50 2.0 × 10⁻³ HVT gD 3.5 × 10⁻³ 7 gIVZV gI 4.2 × 10⁻² HVT gI 7.9 × 10⁻² SVV gI 4.3 × 10⁻¹ 8 gE SHV SA8 gE1.7 × 10⁻⁶ HSV1 gE 1.1 × 10⁻³ BHV1 gE 1.5 × 10⁻² 9 ORF 9 EBV BLRF2 5.7 ×10⁻¹ SR1 no HSV MDV “ORF3” 4.8 × 10⁻⁴ homologue HVT “ORF3” 2.6 × 10⁻¹SR2 US 10 EHV-4 US10 1.2 × 10⁻¹ HSV-1 US10 8.7 × 10⁻¹ EHV-1 US10 8.7 ×10⁻¹

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1. An isolated nucleic acid molecule encoding a polypeptide comprisingthe amino acid sequence of SEQ ID NO:
 11. 2. The isolated nucleic acidmolecule of claim 1, wherein the isolated nucleic acid moleculecomprises the nucleotide sequence of SEQ ID NO:
 10. 3. The isolatednucleic acid molecule of claim 1, wherein the isolated nucleic acidmolecule is cDNA.
 4. An isolated RNA molecule that corresponds to theisolated nucleic acid molecule of claim
 1. 5. The isolated nucleic acidmolecule of claim 1, operatively linked to a regulatory element.
 6. Arecombinant DNA molecule encoding a polypeptide comprising the aminoacid sequence of SEQ ID NO:
 11. 7. The recombinant DNA molecule of claim6, comprising the nucleotide sequence of SEQ ID NO:
 10. 8. Therecombinant DNA molecule of claim 6, wherein the DNA is operativelylinked to a regulatory element.
 9. The recombinant DNA molecule of claim6, wherein the recombinant DNA molecule further comprises DNA encodinginfectious laryngotracheitis virus glycoprotein I.
 10. A host cellcomprising the recombinant DNA molecule of claim
 6. 11. A host cellcomprising the recombinant DNA molecule of claim
 9. 12. A method ofdiagnosing an infection due to infectious laryngotracheitis virus whichcomprises: (a) obtaining a nucleic acid molecule from a suitable bodilyfluid of a subject; (b) contacting the nucleic acid molecule with thelabeled nucleic acid molecule of claim 1 under hybridizing conditions;and (c) determining the presence of the nucleic acid moleculehybridized, the presence of which is indicative of infectiouslaryngotracheitis virus glycoprotein D in the subject, and therebydiagnosing an infection due to infectious laryngotracheitis virus.